Guide rna with chemical modifications

ABSTRACT

The present invention relates to modified guide RNAs and their use in clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems.

CROSS-REFERENCING

This application is a continuation of U.S. application Ser. No.14/757,204, filed on Dec. 3, 2015, and claims the benefit of U.S.Provisional Application No. 62/256,095, filed on Nov. 16, 2015; U.S.Provisional Application No. 62/146,189, filed on Apr. 10, 2015; and U.S.Provisional Application No. 62/087,211, filed on Dec. 3, 2014, all ofwhich arc incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the field of molecular biology. Inparticular, the present invention relates to the dusters of regularlyinterspaced short palindromic repeats (CRISPR) technology.

BACKGROUND OF THE INVENTION

The native prokaryotic CRISPR-Cas system comprises an array of shortrepeats with intervening variable sequences of constant length (i.e.,clusters of regularly interspaced short palindromic repeats, or“CRISPR”), and CRISPR-associated (“Cas”) proteins. The RNA of thetranscribed CRISPR array is processed by a subset of the Cas proteinsinto small guide RNAs, which generally have two components as discussedbelow. There are at least three different systems: Type I, Type II andType III. The enzymes involved in the processing of the RNA into maturecrRNA are different in the 3 systems. In the native prokaryotic system,the guide RNA (“gRNA”) comprises two short, non-coding RNA speciesreferred to as CRISPR RNA (“crRNA”) and trans-acting RNA (“tracrRNA”).In an exemplary system, the gRNA forms a complex with a Cas nuclease.The gRNA:Cas nuclease complex binds a target polynucleotide sequencehaving a protospacer adjacent motif (“PAM”) and a protospacer, which isa sequence complementary to a portion of the gRNA. The recognition andbinding of the target polynucleotide by the gRNA:Cas nuclease complexinduces cleavage of the target polynucleotide. The native CRISPR-Cassystem functions as an immune system in prokaryotes, where gRNA:Casnuclease complexes recognize and silence exogenous genetic elements in amanner analogous to RNAi in eukaryotic organisms, thereby conferringresistance to exogenous genetic elements such as plasmids and phages.

It has been demonstrated that a single-guide RNA (“sgRNA”) can replacethe complex formed between the naturally-existing crRNA and tracrRNA.

Considerations relevant to developing a gRNA, including a sgRNA, includespecificity, stability, and functionality. Specificity refers to theability of a particular gRNA:Cas nuclease complex to bind to and/orcleave a desired target sequence, whereas little or no binding and/orcleavage of polynucleotides different in sequence and/or location fromthe desired target occurs. Thus, specificity refers to minimizingoff-target effects of the gRNA:Cas nuclease complex. Stability refers tothe ability of the gRNA to resist degradation by enzymes, such asnucleases, and other substances that exist in intracellular andextra-cellular environments. Thus, there is a need for providing gRNA,including sgRNA, having increased resistance to nucleolytic degradation,increased binding affinity for the target polynucleotide, and/or reducedoff-target effects while, nonetheless, having gRNA functionality.Further considerations relevant to developing a gRNA includetransferability and immunostimulatory properties. Thus, there is a needfor providing gRNA, including sgRNA, having efficient and titratabletransferability into cells, especially into the nuclei of eukaryoticcells, and having minimal or no immunostimulatory properties in thetransfected cells. Another important consideration for gRNA is toprovide an effective means for delivering it into and maintaining it inthe intended cell, tissue, bodily fluid or organism for a durationsufficient to allow the desired gRNA functionality.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a set of diagrams showing a schematic model of an exemplaryCRISPR-Cas system. The exemplary system shown here is a Type II systemhaving a Cas nuclease. In this particular example, the Cas nuclease isthe Cas9 nuclease. The Cas9 nuclease recognizes a PAM sequence (here,the PAM sequence is a 3-nt sequence of NGG, where N is A, G, C or T, butother PAM sequences are known to exist). The sgRNA includes a guidesequence, a crRNA sequence or segment, and tracrRNA sequence or segment.The guide sequence of the sgRNA hybridizes with the DNA target directlyupstream of the PAM sequence. In the example shown here, Cas9 mediates adouble-stranded break upstream of the PAM sequence (arrows).

FIG. 2A is a diagram showing an exemplary CRISPR-Cas9-mediated cleavageassay.

FIG. 2B is a table showing components and their concentrations for abiochemical cleavage assay used to generate the data in FIG. 4.

FIG. 2C is a diagram showing titration of Streptococcus, pyogenes Cas9nuclease for the biochemical cleavage assay.

FIG. 2D is a diagram showing titration of an exemplary sgRNA for thebiochemical cleavage assay. In this example a sgRNA named kanC1 istargeted to a complementary sequence in the kanamycin resistance gene.

FIG. 3 shows exemplary conditions and procedures for the biochemicalcleavage assay which uses purified components in vitro.

FIG. 4 is a table showing the data obtained using exemplary modifiedguide RNAs m the cleavage assay.

FIG. 5A shows an exemplary guide RNA disclosed in the application.

FIG. 5B shows an exemplary single guide RNA (sgRNA) disclosed in theapplication.

FIG. 6 is a table showing exemplary guide RNAs baying at least twochemical modifications (e.g., a first modification and a secondmodification). Each number represents a modification as indicated andeach “x” indicates the combination of modifications in a guide RNA. Incertain embodiments, the first and second modifications are present on asingle nucleotide. In certain embodiments, the first and secondmodifications are present on separate nucleotides.

FIG. 7 shows exemplary types of guide RNAs having at least threechemical modifications. The lower part of FIG. 7 lists several types ofmodifications. The table in the upper part of FIG. 7 indicates how adouble modification (“double mod,” a combination of two types ofmodifications) can be combined with a single modification (“single mod,”one type of modification). An “x” indicates the presence of thecorresponding double mod and single mod in a guide RNA.

FIGS. 8A and 8B show fluorophore-modified CLTA1 sgRNAs for in vitrotesting. In FIG. 8A, the RNA sequence of a sgRNA for CLTA1 is shown,including a position where a fluorescent dye or a label could beattached to the sgRNA. FIG. 8B shows a structure determined by Xraycrystallograpy of a Cas9:sgRNA complex, as reported in Nishimasu et al.,Cell (2014) 156, 1-15.

FIG. 9A shows CTLA1 sgRNAs modified with 2-thiouridine at certainlocations (positions 3, 9 and 11) in an effort to improve specificityfor the CTLA1 target. FIG: 9B shows that gRNA modified with 2-thioU canincrease target specificity of the gRNAs when off-target sites involveU-G wobble pairing. In particular, the CTLA1_2-thioU+11 had much lowercleavage of the off-target sequence CLTA1 OFF3, which has a T to Cmutation at the 11th position in the 5′ strand.

FIG. 10 shows the guide RNA scaffold secondary structure; displayingnoncovalent binding interactions with amino acids of Cas9, as reportedin Jiang et al., Science (2015) 348:6242, 1477-81.

FIGS. 11A and 11B illustrate experimental results showing that genedisruption in human cell lines, with high frequencies of indels andhomologous recombination (HR), can be-achieved using synthesized andchemically modified sgRNAs disclosed herein, as reported in Hendel etal., Nat. Biotechnol. (2015) 53:9, 985-9.

FIGS. 12A, 12B, 12C and 12D illustrate experimental results showing thatchemically modified sgRNAs as described herein can be used to achievehigh frequencies of gene disruption or targeted genome editing instimulated primary human T cells as well as in CD34+ hematopoietic stemand progenitor cells (HSPCs), as reported in Hendel et al., Nat.Biotechnol. (2015) 33:9, 985-9.

DETAILED DESCRIPTION OF THE INVENTION

This invention is based, at least in part, on an unexpected discoverythat certain chemical modifications to gRNA are tolerated by theCRISPR-Cas system. In particular, certain chemical modificationsbelieved to increase the stability of the gRNA, to alter thethermostability of a gRNA hybridization interaction, and/or to decreasethe off-target effects of Cas:gRNA complexation do not substantiallycompromise the efficacy of Cas:gRNA binding to, nicking of, and/orcleavage of the target polynucleotide. Furthermore, certain chemicalmodifications are believed to provide gRNA, including sgRNA, havingefficient and titratable transferability into cells, especially into thenuclei of eukaryotic cells, and/or having minimal or noimmunostimulatory properties in the transfected cells. Certain chemicalmodifications are believed to provide gRNA, including sgRNA, which canbe effectively delivered into and maintained in the intended cell,tissue, bodily fluid or organism for a duration sufficient to allow thedesired gRNA functionality.

I. Definitions

As used herein, the term “guide RNA” generally refers to an RNA molecule(or a group of RNA molecules collectively) that can bind to a Casprotein and aid in targeting, the Cas protein to a specific locationwithin a target polynucleotide (e.g., a DNA). A guide RNA can comprise acrRNA segment and a tracrRNA segment. As used herein, the term “crRNA”or “crRNA segment” refers to an RNA molecule or portion thereof thatincludes a polynucleotide-targeting guide sequence, a stem sequence,and, optionally, a 5-overhang sequence. As used herein, the term“tracrRNA” or “tracrRNA segment” refers to an RNA molecule or portionthereof that includes a protein-binding segment (e.g., theprotein-binding segment is capable of interacting with aCRISPR-associated protein, such as a Cas9). The term “guide RNA”encompasses a single guide RNA (sgRNA), where the crRNA segment and thetracrRNA segment are located in the same RNA molecule. The term “guideRNA” also encompasses, collectively, a group of two or more RNAmolecules, where the crRNA segment and the tracrRNA segment are locatedin separate RNA molecules.

The term “scaffold” refers to the portions of guide RNA moleculescomprising sequences which are substantially identical or are highlyconserved across natural biological species. Scaffolds include thetracrRNA segment and the portion of the crRNA segment other than thepolynucleotide-targeting guide sequence at or near the 5′ end of thecrRNA segment, excluding any unnatural portions comprising sequences notconserved in native crRNAs and tracrRNAs.

The term “nucleic acid”, “polynucleotide” or “oligonucleotide” refers toa DNA molecule, an RNA molecule, or analogs thereof. As used herein, theterms “nucleic acid”, “polynucleotide” and “oligonucleotide” include,but are not limited to DNA molecules such as cDNA, genomic DNA orsynthetic DNA and RNA molecules such as a guide RNA, messenger RNA orsynthetic RNA. Moreover, as used herein, the terms “nucleic acid” and“polynucleotide” include single-stranded and double-stranded forms.

The term “modification” in the context of an oligonucleotide orpolynucleotide includes but is not limited to (a) end modifications,e.g., 5′ end modifications or 3′ end modifications, (b) nucleobase (or“base”) modifications, including replacement or removal of bases, (c)sugar modifications, including modifications at the 2′, 3′, and/or 4′positions, and (d) backbone modifications, including modification orreplacement of the phosphodiester linkages. The term “modifiednucleotide” generally refers to a nucleotide having a modification tothe chemical structure of one or more of the base, the sugar, and thephosphodiester linkage or backbone portions, including nucleotidephosphates.

The terms “Z” and “P” refer to the nucleotides, nucleobases, ornucleobase analogs developed by Steven Benner and colleagues asdescribed for example in “Artificially expanded genetic informationsystem: a new base pair with an alternative hydrogen bonding pattern”Yang, Z., Hutter, D., Sheng, P., Sismour, A. M. and Benner, S. A. (2006)Nucleic Acids Res., 34, 6095-101, the contents of which is herebyincorporated by reference in its entirety.

The terms “xA”, “xG”, “xC”, “xT”, or “x(A,G,C,T)” and “yA”, “yG”, “yC”,“yT”, or “y(A,G,C,T)” refer to nucleotides, nucleobases, or nucleobaseanalogs as described by Krueger et al in “Synthesis and Properties ofSize-Expanded DNAs: Toward Designed, Functional Genetic Systems”; AndrewT. Krueger, Haige Lu, Alex H. F. Lee, and Eric T. Kool (2007) Acc. Chem.Res., 40, 141-50, the contents of which is hereby incorporated byreference in its entirety.

The term “Unstructured Nucleic Acid” or “UNA” refers to nucleotides,nucleobases, or nucleobase analogs as described in U.S. Pat. No.7,371,580, the contents of which is hereby incorporated by reference inits entirety. An unstructured nucleic acid, or UNA, modification is alsoreferred to as a “pseudo-complementary” nucleotide, nucleobase ornucleobase analog (see e.g., Lahoud et al. (1991) Nucl. Acids Res.,36:10, 3409-19).

The terms “PAGE” and “thioPACE” refer to internucleotide phosphodiesterlinkage analogs containing phosphonoacetate or thiophosphonoacetategroups, respectively. These modifications belong to abroad class ofcompounds comprising phosphonocarboxylate moiety, phosphonocarboxylateester moiety, thiophosphonocarboxylate moiety andthiophosphonocarboxylate ester moiety. These linkages can be describedrespectively by the general formulae P(CR₁R₂)_(n)COOR and(S)—P(CR₁R₂)_(n)COOR wherein n is an integer from 0 to 6 and each of R₁and R₂ is independently selected from the group consisting of H, analkyl and substituted alkyl. Some of these modifications arc describedby Yamada et al. in “Synthesis and Biochemical Evaluation ofPhosphonoformate Oligodeoxyribonucleotides” Christina M. Yamada, DouglasJ. Dellinger and Marvin H. Caruthers (2006) J. Am. Chem. Soc. 128:15,5251-61, the contents of which is hereby incorporated by reference inits entirety.

As used herein, “modification” refers to a chemical moiety, or portionof a chemical structure, which differs from that found in unmodifiedribonucleotides, namely adenosine, guanosine, cytidine, and uridineribonucleotides. The term “modification” may refer to type ofmodification. For example, “same modification” means same type ofmodification, and “the modified nucleotides are the same” means themodified nucleotides have the same type(s) of modification while thebase (A, G, C, U, etc.) may be different. Similarly, a guide RNA with“two modifications” is a guide RNA with two types of modifications,which may or may not be in the same nucleotide, and each type may appearin multiple nucleotides in the guide RNA. Similarly, a guide RNA with“three modifications” is a guide RNA with three types of modifications,which may or may not be in the same nucleotide, and each type may appearin multiple nucleotides.

As used herein, the term “target polynucleotide” or “target” refers to apolynucleotide containing a target nucleic acid sequence. A targetpolynucleotide may be single-stranded or double-stranded, and, incertain embodiments, is double-stranded DNA. In certain embodiments, thetarget polynucleotide is single-stranded RNA. A “target nucleic acidsequence” or “target sequence,” as used herein, means a specificsequence or the complement thereof that one wishes to bind to, nick, orcleave using a CRISPR system.

The term “hybridization” or “hybridizing” refers to a process wherecompletely or partially complementary polynucleotide strands cometogether under suitable hybridization conditions to form adouble-stranded structure or region in which the two constituent strandsare joined by hydrogen bonds. As used herein, the term “partialhybridization” includes where the double-stranded structure or regioncontains one or more bulges or mismatches. Although hydrogen bondstypically form between adenine and thymine or adenine and uracil (A andT or A and U) or cytosine and guanine (C and G), other noncanonical basepairs may form (See e.g., Adams el al, “The Biochemistry of the NucleicAcids, ” 11th ed., 1992). It is contemplated that modified nucleotidesmay form hydrogen bonds that allow or promote hybridization.

The term “cleavage” or “cleaving” refers to breaking of the covalentphosphodiester linkage in the ribosylphosphodiester backbone of apolynucleotide. The terms “cleavage” or “cleaving” encompass bothsingle-stranded breaks and double-stranded breaks. Double-strandedcleavage can occur as a result of two distinct single-stranded cleavageevents. Cleavage can result in the production of either blunt ends orstaggered ends.

The term “CRISPR-associated protein” or “Cas protein” refers to a wildtype Cas protein, a fragment thereof, or a mutant or variant thereof.The term “Cas mutant” or “Cas variant” refers to a protein orpolypeptide derivative of a wild type Cas protein, e.g., a proteinhaving one or more point mutations, insertions, deletions, truncations,a fusion protein, or a combination thereof. In certain embodiments, the“Cas mutant” or “Cas variant” substantially retains the nucleaseactivity of the Cas protein. In certain embodiments, the “Cas mutant” or“Cas variant” is mutated such that one or both nuclease domains areinactive. In certain embodiments, the “Cas mutant” or “Cas variant” hasnuclease activity. In certain embodiments, the “Cas mutant” or “Casvariant” lacks some or all of the nuclease activity of its wild-typecounterpart.

The term “nuclease domain” of a Cas protein refers to the polypeptidesequence or domain within the protein which possesses the catalyticactivity for DNA cleavage. A nuclease domain can be contained in asingle polypeptide chain, or cleavage activity can result from theassociation of two (or more), polypeptides. A single nuclease domain mayconsist of more than one isolated stretch of amino acids within a givenpolypeptide. Examples of these domains include RuvC-like motifs (aminoacids 7-22,759-766 and 982-989 in SEQ ID NO: 1) and HNH motif (aa837-863). Sec Gasiunas et al. (2012) Proc. Natl. Acad. Sci. USA, 109:39,E2579-E2586 and WO2013176772.

A synthetic guide RNA that has “gRNA functionality” is one that has oneor more of the functions of naturally occurring guide RNA, such asassociating with a Cas protein, or a function performed by the guide RNAin association with a Cas protein. In certain embodiments, thefunctionality includes binding a target polynucleotide. In certainembodiments, the functionality includes targeting a Cas protein or agRNA:Cas protein complex to a target polynucleotide. In certainembodiments, the functionality includes nicking a target polynucleotide.In certain embodiments, the functionality includes cleaving a targetpolynucleotide. In certain embodiments, the functionality includesassociating with or binding to a Cas protein. In certain embodiments,the functionality is any other known function of a guide RNA in aCRISPR-Cas system with a Cas protein, including an artificial CRISPR-Cassystem with an engineered Cas protein. In certain embodiments, thefunctionality is any other function of natural guide RNA. The syntheticguide RNA may have gRNA functionality to a greater or lesser extent thana naturally occurring guide RNA. In certain embodiments, a syntheticguide RNA may have greater functionality as to one property and lesserfunctionality as to another property in comparison to a similarnaturally occurring guide RNA.

A “Cas protein having a single-strand nicking activity” refers to a Casprotein, including a Cas mutant or Cas variant, that has reduced abilityto cleave one of two strands of a dsDNA as compared to a wild type Casprotein. For example, in certain embodiments, a Cas protein having asingle-strand nicking activity has a mutation (e.g., amino acidsubstitution) that reduces the function of the RuvC domain (or the HNHdomain) and as a result reduces the ability to cleave one strand of thetarget DNA. Examples of such variants include the D10A, H839A/H840A,and/or N863A substitutions in S. pyogenes Cas9, and also include thesame or similar substitutions at equivalent sites in Cas9 enzymes ofother species.

As used herein, the term “portion” or “fragment” of a sequence refers toany portion of the sequence (e.g., a nucleotide subsequence or an aminoacid subsequence) that is smaller than the complete sequence. Portionsof polynucleotides can be any length, for example, at least 5, 10, 15,20, 25, 30, 40, 50, 75, 100, 150, 200, 300 or 500 or more nucleotides inlength. A portion of a guide sequence can be about 50%, 40%, 30%, 20%,10% of the guide sequence, e.g., one-third of the guide sequence orshorter, e.g., 7, 6, 5, 4, 3, or 2 nucleotides in length.

The term “derived from” in the context of a molecule refers to amolecule isolated or made using a parent molecule or information fromthat parent molecule. For example, a Cas9 single mutant nickase and aCas9 double mutant null-nuclease are derived from a wild-type Cas9protein.

The term “substantially identical” in the context of two or morepolynucleotides (or two or more polypeptides) refers to sequences orsubsequences that have at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, about 90-95%, at least about 95%, atleast about 98%, at least about 99% or more nucleotide (or amino acid)sequence identity, when compared and aligned for maximum correspondenceusing a sequence comparison algorithm or by visual inspection.Preferably, the “substantial identity” between polynucleotides existsover a region of the polynucleotide at least about 50 nucleotides inlength, at least about 100 nucleotides in length, at least about 200nucleotides in length, at least about 300 nucleotides in lengthy atleast about 500 nucleotides in length, or over the entire length of thepolynucleotide. Preferably, the “substantial identity” betweenpolypeptides exists over a region of the polypeptide at least about 50amino acid residues in length, at least about 100 amino acid residues inlength, or over the entire length of the polypeptide.

As disclosed herein, a number of ranges of values are provided. It isunderstood that each intervening value, to the tenth of the unit of thelower limit, between the upper and lower limits of that range is alsospecifically contemplated. Each smaller range or intervening valueencompassed by a stated range is also specifically contemplated. Theterm “about” generally refers to plus or minus 10% of the indicatednumber. For example, “about 10%” may indicate a range of 9% to 11%, and“about 20” may mean from 18-22. Other meanings of “about” may beapparent from the context, such as rounding off, so, for example “about1” may also mean from 0.5 to 1.4.

II. CRISPR-Mediated Sequence-Specific Binding and/or Cleavage

Shown in FIG. 1 is a diagram of CRISPR-Cas9-mediated sequence-specificcleavage of DNA. The guide RNA is depicted as sgRNA with an exemplary20-nucleotide (20-nt) guide sequence (other guide sequences may be, forexample, from about 15 to about 30 nts in length) within the 5′ domain,an internally positioned base-paired stem, and a 3′ domain. The guidesequence is complementary to an exemplary 20-nt target sequence in a DNAtarget. The stem corresponds to a repeat sequence in crRNA and iscomplementary to a sequence in the tracrRNA. The 3′ domain of the guideRNA corresponds to the 3′ domain of the tracrRNA that binds a Cas9nuclease. The Cas9:guide RNA complex binds and cleaves a target DNAsequence or protospacer directly upstream of a PAM sequence recognizedby Cas9. In FIG. 1, a 3-nt PAM sequence is exemplified; however otherPAM sequences, including 4-nt and 5-nt PAM sequences are known. In thesystem exemplified in FIG. 1, both strands of the target sequence in DNAare cleaved by Cas9 at the sites indicated by arrows.

III. Guide RNAs

In at least one aspect, the present invention comprises a chemicallymodified guide RNA that has guide RNA functionality. A guide RNA thatcomprises any nucleotide other than the four canonical ribonucleotides,namely A, C, G, and U, whether unnatural or natural (e.g., apseudouridine, inosine or a deoxynucleotide), is a chemically modifiedguide RNA. Likewise a guide RNA that comprises any backbone orinternucleotide linkage other than a natural phosphodiesterinternucleotide linkage possesses a chemical modification and thereforeis a chemically modified guide RNA. In certain embodiments, the retainedfunctionality includes binding a Cas protein. In certain embodiments,the retained functionality includes binding a target polynucleotide. Incertain embodiments, the retained functionality includes targeting a Casprotein or a gRNA:Cas protein complex to a target polynucleotide. Incurtain embodiments, the retained functionality includes nicking atarget polynucleotide by a gRNA:Cas protein complex. In certainembodiments, the retained functionality includes cleaving a targetpolynucleotide by a gRNA:Cas protein complex. In certain embodiments,the retained functionality is any other known function of a guide RNA ina CRISPR-Cas system with a Cas protein, including an artificialCRISPR-Cas system with an engineered Cas protein. In certainembodiments, the retained functionality is any other function of anatural guide RNA.

A. Exemplary Modifications

In certain embodiments, a nucleotide sugar modification incorporatedinto the guide RNA is selected from the group consisting of2′-O-C₁₋₄alkyl such as 2′-O-methyl (2′-OMe), 2′-deoxy (2′-H),2′-O-C₁₋₃alkyl-O-Cu₁₋₃alkyl such as 2′-methoxyethyl (“2′-MOE”),2′-fluoro (“2′-F”), 2′-amino (“2′-NH₂”), 2′-arabinosyl (“2′-arabino”)nucleotide, 2′-F-arabinosyl (“2′-F-arabino”) nucleotide, 2′-lockednucleic acid (“LNA”) nucleotide, 2′-unlocked nucleic acid (“ULNA”)nucleotide, a sugar in L form (“L-sugar”), and 4′-thioribosylnucleotide. In certain embodiments, an internucleotide linkagemodification incorporated into the guide RNA is selected from the groupconsisting of: phosphorothioate “P(S)” (P(S)), phosphonocarboxylate(P(CH₂)_(n)COOR) such as phosphonoacetate “PACE” (P(CH₂COO⁻))thiophosphonocarboxylate ((S)P(CH₂)_(n)COOR) such asthiophosphonoacetate “thioPACE” ((S)P(CH₂COO⁻)), alkylphosphonate(P(C₁₋₃alkyl) such as methylphosphonate —P(CH₃), boranophosphonate(P(BH₃)), and phosphorodithioate (P(S)₂).

In certain embodiments, a nucleobase (“base”) modification incorporatedinto the guide RNA is selected from the group consisting of:2-thiouracil (“2-thioU”), 2-thiocytosine (“2-thioC”), 4-thiouracil(“4-thioU”), 6-thioguanine (“6-thioG”), 2-aminoadenine (“2-aminoA”),2-aminopurine, pseudouracil, hypoxanthine, 7-deazaguanine,7-deaza-8-azaguanine, 7-deazaadenine, 7-deaza-8-azaadenine,5-methylcytosine (“5-methylC”), 5-methyluracil (“5-methylU”),5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5,6-dehydrouracil,5-propynylcytosine, 5-propynyluracil, 5-ethynylcytosine,5-ethynyluracil, 5-allyluracil (“5-allylU”), 5-allylcytosine(“5-allylC”), 5-aminoallyluracil (“5-aminoallylU”),5-aminoallyl-cytosine (“5-aminoallylC”), an abasic nucleotide, Z base, Pbase, Unstructured Nucleic Acid (“UNA”), isoguanine (“isoG”),isocytosine (“isoC”) [as described in “Enzymatic Incorporation of a NewBase pair into DNA and RNA Extends the Genetic Alphabet.”Piccirilli, J.A.; Krauch, T.; Moroncy, S. E.; Benner, S. A. (1990) Nature, 343, 33],5-methyl-2-pyrimidine [as described in Rappaport, H. P. (1993)Biochemistry, 32, 3047], x(A,G,C,T) and y(A,G,C,T).

In certain embodiments, one or more isotopic modifications areintroduced on the nucleotide sugar, the nucleobase, the phosphodiesterlinkage and/or the nucleotide phosphates. Such modifications includenucleotides comprising one or more ¹⁵N, ¹³C, ¹⁴C, Deuterium, ³H, ³²P,¹²⁵I, ¹³¹I atoms or other atoms or elements used as tracers.

In certain embodiments, an “end” modification incorporated into theguide RNA is selected from the group consisting of: PEG(polyethyleneglycol), hydrocarbon linkers (including: heteroatom(O,S,N)-substituted hydrocarbon spacers; halo-substituted hydrocarbonspacers; keto-, carboxyl-, amido-, thionyl-, carbamoyl-,thionocarbamaoyl-containing hydrocarbon spacers), spermine linkers, dyesincluding fluorescent dyes-(for example fluoresceins, rhodamines,cyanines) attached to linkers such as for example 6-fluorescein-hexyl,quenchers (for example dabcyl, BHQ) and other labels (for examplebiotin, digoxigenin, acridine, streptavidin, avidin, peptides and/orproteins). In certain embodiments, an “end” modification comprises aconjugation (or ligation) of the guide RNA to another moleculecomprising an oligonucleotide (comprising deoxynucleotides and/orribonucleotides), a peptide, a protein, a sugar, an oligosaccharide, asteroid, a lipid, a folic acid, a vitamin and/or other molecule. Incertain embodiments, an “end” modification incorporated into the guideRNA is located internally in the guide RNA sequence via a linker such asfor example 2-(4-butylamidofluorescein)propane-1,3-diolbis(phosphodiester) linker (depicted below), which is incorporated as aphosphodiester linkage and can be incorporated anywhere between twonucleotides in the guide RNA.

2-(4-butylamidofluorescein)propane-1,3-diol bis(phosphodiester) linker

Other linkers include for example by way of illustration, but are notlimited to:

2-(3-(dye-amido)propanamido)propane-1,3-diol bis(phosphodiester) linker

In certain embodiments, the end modification comprises a terminalfunctional group such as an amine, a thiol (or sulfhydryl), a hydroxyl,a carboxyl, carbonyl, thionyl, thiocarbonyl, a carbamoyl, athiocarbamoyl, a phoshoryl, an alkene, an alkyne, an halogen or afunctional group-terminated linker, either of which can be subsequentlyconjugated to a desired moiety, for example a fluorescent dye or anon-fluorescent label or tag or any other molecule such as for examplean oligonucleotide (comprising deoxynucleotides and/or ribonucleotides,including an aptamer), an amino acid, a peptide, a protein, a sugar, anoligosaccharide, a steroid, a lipid, a folic acid, a vitamin. Theconjugation employs standard chemistry well-known in the art, includingbut not limited to coupling via N-hydroxysuccinimide, isothiocyanate;DCC (or DCI), and/or any other standart method as described in“Bioconjugate Techniques” by Greg T. Hermanson, Publisher EslsevierScience, 3rd ed. (2013), the contents of which are incorporated hereinby reference in their entireties.

In certain embodiments, the label or dye is attached or conjugated to amodified nucleotide in the gRNA. The conjugation of a fluorescent dye orother moiety such as a non-fluorescent label or tag (for example biotin,avidin, streptavidin, or moiety containing an isotopic label such as¹⁵N, ¹³C, ¹⁴C, Deuterium, ³H, ³²P, ¹²⁵I and the like) or any othermolecule such as for example an oligonucleotide (comprisingdeoxynucleotides and/or ribonucleotides including an aptamer), an aminoacid, a peptide, a protein, a sugar, an oligosaccharide, a steroid, alipid, a folic acid, a vitamin or other molecule can be effectuatedusing the so-called “click” chemistry or the so-called “squarate”conjugation chemistry. The “click” chemistry refers to the [3+2]cycloaddition of an alkyne moiety with an azide moiety, leading to atriazolo linkage between the two moieties as shown in the followingscheme:

as described for example in El-Sagheer, A. H and Brown, T. “Clickchemistry with DNA”, Chem. Soc. Rev., 2010, 39, 1388-1405 and Mojibul,H. M. and XiaoHua, P., DNA-associated click chemistry, Sci. China Chem.,2014, 57:2, 215-31, the contents of which are hereby incorporated byreference in their entirety.

In certain embodiments, the conjugation can be effectuated byalternative cycloaddition such as Diels-Alder [4+2] cycloaddition of aπ-conjugatcd diene moiety with an alkene moiety.

The “squarate” conjugation chemistry links two moieties each having anamine via a squarate derivative to result in a squarate conjugate thatcontains a squarate moiety (see e.g., Tietze et al. (1991) Chem. Ber.,124, 1215-21, the contents of which arc hereby incorporated by referencein their entirety). For example, a fluorescein containing a linker amineis conjugated to an oligoribonucleotide containing an amine through asquarate linker as described in the scheme below. An example of thesquarate linker is depicted in the following scheme:

In certain, embodiments, a chemical modification incorporated into theguide RNA is selected from the group consisting of 2′-O-C₁₋₄alkyl, 2′-H,2′-O-C₁₋₃alkyl-O—C₁₋₃alkyl, 2′-F, 2′-NH₂, 2′-arabino, 2′-F-arabino,4′-thioribosyl, 2-thioU, 2-thioC, 4-thioU, 6-thioG, 2-aminoA,2-aminopurine, pseudouracil, hypoxanthine, 7-deazaguanine,7-deaza-8-azaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 5-methylC,5-methylU, 5-hydroxymethylcytosine, 5-hydroxymethyluracil,5,6-dehydrouracil, 5-propynylcytosine, 5-propynyluracil,5-ethynylcytosine, 5-ethynyluracil, 5-allylU, 5-allylC,5-aminoallyl-uracil, 5-aminoallyl-cytosine, an abasic nucleotide(“abN”), Z, P, UNA, isoC, isoG, 5-methyl-pyrimidine, x(A,G,C,T) andy(A,G,C,T), a phosphorothioate internucleotide linkage, aphosphonoacetate internucleotide linkage, a thiophosphonoacetateinternucleotide linkage, a methylphosphonate internucleotide linkage, aboranophosphonate internucleotide linkage, a phosphorodithioateinternucleotide linkage, 4-thioribosyl nucleotide, a locked nucleic acid(“LNA”) nucleotide, an unlocked nucleic acid (“ULNA”) nucleotide, analkyl spacer, a heteroalkyl (N, O, S) spacer, a 5′- and/or 3-alkylterminated nucleotide, a Unicap, a 5′-terminal cap known from nature, anxRNA base (analogous to “xDNA” base), an yRNA base (analogous to “yDNA”base), a PEG substituent, or a conjugated linker to a dye ornon-fluorescent label (or tag) or other moiety as described above.Exemplary modified nucleotides are also depicted in Table 2.

TABLE 2 Exemplary modified nucleotides contained in a synthetic guidesequence.

# R₁ R₂ B A1 OH OH uridine A2 OMe OH uridine A3 F OH uridine A4 Cl OHuridine A5 Br OH uridine A6 I OH uridine A7 NH₂ OH uridine A8 H OHuridine A9 OH phosphodiester uridine A10 OMe phosphodiester uridine A11F phosphodiester uridine A12 Cl phosphodiester uridine A13 Brphosphodiester uridine A14 I phosphodiester uridine A15 NH₂phosphodiester uridine A16 H phosphodiester uridine A17 OHphosphonoacetate uridine A18 OMe phosphonoacetate uridine A19 Fphosphonoacetate uridine A20 Cl phosphonoacetate uridine A21 Brphosphonoacetate uridine A22 I phosphonoacetate uridine A23 NH₂phosphonoacetate uridine A24 H phosphonoacetate uridine A25 OHthiophosphonoacetate uridine A26 OMe thiophosphonoacetate uridine A27 Fthiophosphonoacetate uridine A28 Cl thiophosphonoacetate uridine A29 Brthiophosphonoacetate uridine A30 I thiophosphonoacetate uridine A31 NH₂thiophosphonoacetate uridine A32 H thiophosphonoacetate uridine A33 OHphosphorothioate uridine A34 OMe phosphorothioate uridine A35 Fphosphorothioate uridine A36 Cl phosphorothioate uridine A37 Brphosphorothioate uridine A38 I phosphorothioate uridine A39 NH₂phosphorothioate uridine A40 H phosphorothioate uridine A41 OHphosphorodithioate uridine A42 OMe phosphorodithioate uridine A43 Fphosphorodithioate uridine A44 Cl phosphorodithioate uridine A45 Brphosphorodithioate uridine A46 I phosphorodithioate uridine A47 NH₂phosphorodithioate uridine A48 H phosphorodithioate uridine A49 OHmethylphosphonate uridine A50 OMe methylphosphonate uridine A51 Fmethylphosphonate uridine A52 Cl methylphosphonate uridine A53 Brmethylphosphonate uridine A54 I methylphosphonate uridine A55 NH₂methylphosphonate uridine A56 H methylphosphonate uridine A57 OHboranophosphonate uridine A58 OMe boranophosphonate uridine A59 Fboranophosphonate uridine A60 Cl boranophosphonate uridine A61 Brboranophosphonate uridine A62 I boranophosphonate uridine A63 NH₂boranophosphonate uridine A64 H boranophosphonate uridine B1 OH OHadenosine B2 OMe OH adenosine B3 F OH adenosine B4 Cl OH adenosine B5 BrOH adenosine B6 I OH adenosine B7 NH₂ OH adenosine B8 H OH adenosine B9OH phosphodiester adenosine B10 OMe phosphodiester adenosine B11 Fphosphodiester adenosine B12 Cl phosphodiester adenosine B13 Brphosphodiester adenosine B14 I phosphodiester adenosine B15 NH₂phosphodiester adenosine B16 H phosphodiester adenosine B17 OHphosphonoacetate adenosine B18 OMe phosphonoacetate adenosine B19 Fphosphonoacetate adenosine B20 Cl phosphonoacetate adenosine B21 Brphosphonoacetate adenosine B22 I phosphonoacetate adenosine B23 NH₂phosphonoacetate adenosine B24 H phosphonoacetate adenosine B25 OHthiophosphonoacetate adenosine B26 OMe thiophosphonoacetate adenosineB27 F thiophosphonoacetate adenosine B28 Cl thiophosphonoacetateadenosine B29 Br thiophosphonoacetate adenosine B30 Ithiophosphonoacetate adenosine B31 NH₂ thiophosphonoacetate adenosineB32 H thiophosphonoacetate adenosine B33 OH phosphorothioate adenosineB34 OMe phosphorothioate adenosine B35 F phosphorothioate adenosine B36Cl phosphorothioate adenosine B37 Br phosphorothioate adenosine B38 Iphosphorothioate adenosine B39 NH₂ phosphorothioate adenosine B40 Hphosphorothioate adenosine B41 OH phosphorodithioate adenosine B42 OMephosphorodithioate adenosine B43 F phosphorodithioate adenosine B44 Clphosphorodithioate adenosine B45 Br phosphorodithioate adenosine B46 Iphosphorodithioate adenosine B47 NH₂ phosphorodithioate adenosine B48 Hphosphorodithioate adenosine B49 OH methylphosphonate adenosine B50 OMemethylphosphonate adenosine B51 F methylphosphonate adenosine B52 Clmethylphosphonate adenosine B53 Br methylphosphonate adenosine B54 Imethylphosphonate adenosine B55 NH₂ methylphosphonate adenosine B56 Hmethylphosphonate adenosine B57 OH boranophosphonate adenosine B58 OMeboranophosphonate adenosine B59 F boranophosphonate adenosine B60 Clboranophosphonate adenosine B61 Br boranophosphonate adenosine B62 Iboranophosphonate adenosine B63 NH₂ boranophosphonate adenosine B64 Hboranophosphonate adenosine C1 OH OH cytidine C2 OMe OH cytidine C3 F OHcytidine C4 Cl OH cytidine C5 Br OH cytidine C6 I OH cytidine C7 NH₂ OHcytidine C8 H OH cytidine C9 OH phosphodiester cytidine C10 OMephosphodiester cytidine C11 F phosphodiester cytidine C12 Clphosphodiester cytidine C13 Br phosphodiester cytidine C14 Iphosphodiester cytidine C15 NH₂ phosphodiester cytidine C16 Hphosphodiester cytidine C17 OH phosphonoacetate cytidine C18 OMephosphonoacetate cytidine C19 F phosphonoacetate cytidine C20 Clphosphonoacetate cytidine C21 Br phosphonoacetate cytidine C22 Iphosphonoacetate cytidine C23 NH₂ phosphonoacetate cytidine C24 Hphosphonoacetate cytidine C25 OH thiophosphonoacetate cytidine C26 OMethiophosphonoacetate cytidine C27 F thiophosphonoacetate cytidine C28 Clthiophosphonoacetate cytidine C29 Br thiophosphonoacetate cytidine C30 Ithiophosphonoacetate cytidine C31 NH₂ thiophosphonoacetate cytidine C32H thiophosphonoacetate cytidine C33 OH phosphorothioate cytidine C34 OMephosphorothioate cytidine C35 F phosphorothioate cytidine C36 Clphosphorothioate cytidine C37 Br phosphorothioate cytidine C38 Iphosphorothioate cytidine C39 NH₂ phosphorothioate cytidine C40 Hphosphorothioate cytidine C41 OH phosphorodithioate cytidine C42 OMephosphorodithioate cytidine C43 F phosphorodithioate cytidine C44 Clphosphorodithioate cytidine C45 Br phosphorodithioate cytidine C46 Iphosphorodithioate cytidine C47 NH₂ phosphorodithioate cytidine C48 Hphosphorodithioate cytidine C49 OH methylphosphonate cytidine C50 OMemethylphosphonate cytidine C51 F methylphosphonate cytidine C52 Clmethylphosphonate cytidine C53 Br methylphosphonate cytidine C54 Imethylphosphonate cytidine C55 NH₂ methylphosphonate cytidine C56 Hmethylphosphonate cytidine C57 OH boranophosphonate cytidine C58 OMeboranophosphonate cytidine C59 F boranophosphonate cytidine C60 Clboranophosphonate cytidine C61 Br boranophosphonate cytidine C62 Iboranophosphonate cytidine C63 NH₂ boranophosphonate cytidine C64 Hboranophosphonate cytidine D1 OH OH guanosine D2 OMe OH guanosine D3 FOH guanosine D4 Cl OH guanosine D5 Br OH guanosine D6 I OH guanosine D7NH₂ OH guanosine D8 H OH guanosine D9 OH phosphodiester guanosine D10OMe phosphodiester guanosine D11 F phosphodiester guanosine D12 Clphosphodiester guanosine D13 Br phosphodiester guanosine D14 Iphosphodiester guanosine D15 NH₂ phosphodiester guanosine D16 Hphosphodiester guanosine D17 OH phosphonoacetate guanosine D18 OMephosphonoacetate guanosine D19 F phosphonoacetate guanosine D20 Clphosphonoacetate guanosine D21 Br phosphonoacetate guanosine D22 Iphosphonoacetate guanosine D23 NH₂ phosphonoacetate guanosine D24 Hphosphonoacetate guanosine D25 OH thiophosphonoacetate guanosine D26 OMethiophosphonoacetate guanosine D27 F thiophosphonoacetate guanosine D28Cl thiophosphonoacetate guanosine D29 Br thiophosphonoacetate guanosineD30 I thiophosphonoacetate guanosine D31 NH₂ thiophosphonoacetateguanosine D32 H thiophosphonoacetate guanosine D33 OH phosphorothioateguanosine D34 OMe phosphorothioate guanosine D35 F phosphorothioateguanosine D36 Cl phosphorothioate guanosine D37 Br phosphorothioateguanosine D38 I phosphorothioate guanosine D39 NH₂ phosphorothioateguanosine D40 H phosphorothioate guanosine D41 OH phosphorodithioateguanosine D42 OMe phosphorodithioate guanosine D43 F phosphorodithioateguanosine D44 Cl phosphorodithioate guanosine D45 Br phosphorodithioateguanosine D46 I phosphorodithioate guanosine D47 NH₂ phosphorodithioateguanosine D48 H phosphorodithioate guanosine D49 OH methylphosphonateguanosine D50 OMe methylphosphonate guanosine D51 F methylphosphonateguanosine D52 Cl methylphosphonate guanosine D53 Br methylphosphonateguanosine D54 I methylphosphonate guanosine D55 NH₂ methylphosphonateguanosine D56 H methylphosphonate guanosine D57 OH boranophosphonateguanosine D58 OMe boranophosphonate guanosine D59 F boranophosphonateguanosine D60 Cl boranophosphonate guanosine D61 Br boranophosphonateguanosine D62 I boranophosphonate guanosine D63 NH₂ boranophosphonateguanosine D64 H boranophosphonate guanosine E1 OH OH 2-thiouridine E2OMe OH 2-thiouridine E3 F OH 2-thiouridine E4 Cl OH 2-thiouridine E5 BrOH 2-thiouridine E6 I OH 2-thiouridine E7 NH₂ OH 2-thiouridine E8 H OH2-thiouridine E9 OH phosphodiester 2-thiouridine E10 OMe phosphodiester2-thiouridine E11 F phosphodiester 2-thiouridine E12 Cl phosphodiester2-thiouridine E13 Br phosphodiester 2-thiouridine E14 I phosphodiester2-thiouridine E15 NH₂ phosphodiester 2-thiouridine E16 H phosphodiester2-thiouridine E17 OH phosphonoacetate 2-thiouridine E18 OMephosphonoacetate 2-thiouridine E19 F phosphonoacetate 2-thiouridine E20Cl phosphonoacetate 2-thiouridine E21 Br phosphonoacetate 2-thiouridineE22 I phosphonoacetate 2-thiouridine E23 NH₂ phosphonoacetate2-thiouridine E24 H phosphonoacetate 2-thiouridine E25 OHthiophosphonoacetate 2-thiouridine E26 OMe thiophosphonoacetate2-thiouridine E27 F thiophosphonoacetate 2-thiouridine E28 Clthiophosphonoacetate 2-thiouridine E29 Br thiophosphonoacetate2-thiouridine E30 I thiophosphonoacetate 2-thiouridine E31 NH₂thiophosphonoacetate 2-thiouridine E32 H thiophosphonoacetate2-thiouridine E33 OH phosphorothioate 2-thiouridine E34 OMephosphorothioate 2-thiouridine E35 F phosphorothioate 2-thiouridine E36Cl phosphorothioate 2-thiouridine E37 Br phosphorothioate 2-thiouridineE38 I phosphorothioate 2-thiouridine E39 NH₂ phosphorothioate2-thiouridine E40 H phosphorothioate 2-thiouridine E41 OHphosphorodithioate 2-thiouridine E42 OMe phosphorodithioate2-thiouridine E43 F phosphorodithioate 2-thiouridine E44 Clphosphorodithioate 2-thiouridine E45 Br phosphorodithioate 2-thiouridineE46 I phosphorodithioate 2-thiouridine E47 NH₂ phosphorodithioate2-thiouridine E48 H phosphorodithioate 2-thiouridine E49 OHmethylphosphonate 2-thiouridine E50 OMe methylphosphonate 2-thiouridineE51 F methylphosphonate 2-thiouridine E52 Cl methylphosphonate2-thiouridine E53 Br methylphosphonate 2-thiouridine E54 Imethylphosphonate 2-thiouridine E55 NH₂ methylphosphonate 2-thiouridineE56 H methylphosphonate 2-thiouridine E57 OH boranophosphonate2-thiouridine E58 OMe boranophosphonate 2-thiouridine E59 Fboranophosphonate 2-thiouridine E60 Cl boranophosphonate 2-thiouridineE61 Br boranophosphonate 2-thiouridine E62 I boranophosphonate2-thiouridine E63 NH₂ boranophosphonate 2-thiouridine E64 Hboranophosphonate 2-thiouridine F1 OH OH 4-thiouridine F2 OMe OH4-thiouridine F3 F OH 4-thiouridine F4 Cl OH 4-thiouridine F5 Br OH4-thiouridine F6 I OH 4-thiouridine F7 NH₂ OH 4-thiouridine F8 H OH4-thiouridine F9 OH phosphodiester 4-thiouridine F10 OMe phosphodiester4-thiouridine F11 F phosphodiester 4-thiouridine F12 Cl phosphodiester4-thiouridine F13 Br phosphodiester 4-thiouridine F14 I phosphodiester4-thiouridine F15 NH₂ phosphodiester 4-thiouridine F16 H phosphodiester4-thiouridine F17 OH phosphonoacetate 4-thiouridine F18 OMephosphonoacetate 4-thiouridine F19 F phosphonoacetate 4-thiouridine F20Cl phosphonoacetate 4-thiouridine F21 Br phosphonoacetate 4-thiouridineF22 I phosphonoacetate 4-thiouridine F23 NH₂ phosphonoacetate4-thiouridine F24 H phosphonoacetate 4-thiouridine F25 OHthiophosphonoacetate 4-thiouridine F26 OMe thiophosphonoacetate4-thiouridine F27 F thiophosphonoacetate 4-thiouridine F28 Clthiophosphonoacetate 4-thiouridine F29 Br thiophosphonoacetate4-thiouridine F30 I thiophosphonoacetate 4-thiouridine F31 NH₂thiophosphonoacetate 4-thiouridine F32 H thiophosphonoacetate4-thiouridine F33 OH phosphorothioate 4-thiouridine F34 OMephosphorothioate 4-thiouridine F35 F phosphorothioate 4-thiouridine F36Cl phosphorothioate 4-thiouridine F37 Br phosphorothioate 4-thiouridineF38 I phosphorothioate 4-thiouridine F39 NH₂ phosphorothioate4-thiouridine F40 H phosphorothioate 4-thiouridine F41 OHphosphorodithioate 4-thiouridine F42 OMe phosphorodithioate4-thiouridine F43 F phosphorodithioate 4-thiouridine F44 Clphosphorodithioate 4-thiouridine F45 Br phosphorodithioate 4-thiouridineF46 I phosphorodithioate 4-thiouridine F47 NH₂ phosphorodithioate4-thiouridine F48 H phosphorodithioate 4-thiouridine F49 OHmethylphosphonate 4-thiouridine F50 OMe methylphosphonate 4-thiouridineF51 F methylphosphonate 4-thiouridine F52 Cl methylphosphonate4-thiouridine F53 Br methylphosphonate 4-thiouridine F54 Imethylphosphonate 4-thiouridine F55 NH₂ methylphosphonate 4-thiouridineF56 H methylphosphonate 4-thiouridine F57 OH boranophosphonate4-thiouridine F58 OMe boranophosphonate 4-thiouridine F59 Fboranophosphonate 4-thiouridine F60 Cl boranophosphonate 4-thiouridineF61 Br boranophosphonate 4-thiouridine F62 I boranophosphonate4-thiouridine F63 NH₂ boranophosphonate 4-thiouridine F64 Hboranophosphonate 4-thiouridine G1 OH OH 2-aminoadenosine G2 OMe OH2-aminoadenosine G3 F OH 2-aminoadenosine G4 Cl OH 2-aminoadenosine G5Br OH 2-aminoadenosine G6 I OH 2-aminoadenosine G7 NH₂ OH2-aminoadenosine G8 H OH 2-aminoadenosine G9 OH phosphodiester2-aminoadenosine G10 OMe phosphodiester 2-aminoadenosine G11 Fphosphodiester 2-aminoadenosine G12 Cl phosphodiester 2-aminoadenosineG13 Br phosphodiester 2-aminoadenosine G14 I phosphodiester2-aminoadenosine G15 NH₂ phosphodiester 2-aminoadenosine G16 Hphosphodiester 2-aminoadenosine G17 OH phosphonoacetate 2-aminoadenosineG18 OMe phosphonoacetate 2-aminoadenosine G19 F phosphonoacetate2-aminoadenosine G20 Cl phosphonoacetate 2-aminoadenosine G21 Brphosphonoacetate 2-aminoadenosine G22 I phosphonoacetate2-aminoadenosine G23 NH₂ phosphonoacetate 2-aminoadenosine G24 Hphosphonoacetate 2-aminoadenosine G25 OH thiophosphonoacetate2-aminoadenosine G26 OMe thiophosphonoacetate 2-aminoadenosine G27 Fthiophosphonoacetate 2-aminoadenosine G28 Cl thiophosphonoacetate2-aminoadenosine G29 Br thiophosphonoacetate 2-aminoadenosine G30 Ithiophosphonoacetate 2-aminoadenosine G31 NH₂ thiophosphonoacetate2-aminoadenosine G32 H thiophosphonoacetate 2-aminoadenosine G33 OHphosphorothioate 2-aminoadenosine G34 OMe phosphorothioate2-aminoadenosine G35 F phosphorothioate 2-aminoadenosine G36 Clphosphorothioate 2-aminoadenosine G37 Br phosphorothioate2-aminoadenosine G38 I phosphorothioate 2-aminoadenosine G39 NH₂phosphorothioate 2-aminoadenosine G40 H phosphorothioate2-aminoadenosine G41 OH phosphorodithioate 2-aminoadenosine G42 OMephosphorodithioate 2-aminoadenosine G43 F phosphorodithioate2-aminoadenosine G44 Cl phosphorodithioate 2-aminoadenosine G45 Brphosphorodithioate 2-aminoadenosine G46 I phosphorodithioate2-aminoadenosine G47 NH₂ phosphorodithioate 2-aminoadenosine G48 Hphosphorodithioate 2-aminoadenosine G49 OH methylphosphonate2-aminoadenosine G50 OMe methylphosphonate 2-aminoadenosine G51 Fmethylphosphonate 2-aminoadenosine G52 Cl methylphosphonate2-aminoadenosine G53 Br methylphosphonate 2-aminoadenosine G54 Imethylphosphonate 2-aminoadenosine G55 NH₂ methylphosphonate2-aminoadenosine G56 H methylphosphonate 2-aminoadenosine G57 OHboranophosphonate 2-aminoadenosine G58 OMe boranophosphonate2-aminoadenosine G59 F boranophosphonate 2-aminoadenosine G60 Clboranophosphonate 2-aminoadenosine G61 Br boranophosphonate2-aminoadenosine G62 I boranophosphonate 2-aminoadenosine G63 NH₂boranophosphonate 2-aminoadenosine G64 H boranophosphonate2-aminoadenosine H1 OH OH 7-deazaguanosine H2 OMe OH 7-deazaguanosine H3F OH 7-deazaguanosine H4 Cl OH 7-deazaguanosine H5 Br OH7-deazaguanosine H6 I OH 7-deazaguanosine H7 NH₂ OH 7-deazaguanosine H8H OH 7-deazaguanosine H9 OH phosphodiester 7-deazaguanosine H10 OMephosphodiester 7-deazaguanosine H11 F phosphodiester 7-deazaguanosineH12 Cl phosphodiester 7-deazaguanosine H13 Br phosphodiester7-deazaguanosine H14 I phosphodiester 7-deazaguanosine H15 NH₂phosphodiester 7-deazaguanosine H16 H phosphodiester 7-deazaguanosineH17 OH phosphonoacetate 7-deazaguanosine H18 OMe phosphonoacetate7-deazaguanosine H19 F phosphonoacetate 7-deazaguanosine H20 Clphosphonoacetate 7-deazaguanosine H21 Br phosphonoacetate7-deazaguanosine H22 I phosphonoacetate 7-deazaguanosine H23 NH₂phosphonoacetate 7-deazaguanosine H24 H phosphonoacetate7-deazaguanosine H25 OH thiophosphonoacetate 7-deazaguanosine H26 OMethiophosphonoacetate 7-deazaguanosine H27 F thiophosphonoacetate7-deazaguanosine H28 Cl thiophosphonoacetate 7-deazaguanosine H29 Brthiophosphonoacetate 7-deazaguanosine H30 I thiophosphonoacetate7-deazaguanosine H31 NH₂ thiophosphonoacetate 7-deazaguanosine H32 Hthiophosphonoacetate 7-deazaguanosine H33 OH phosphorothioate7-deazaguanosine H34 OMe phosphorothioate 7-deazaguanosine H35 Fphosphorothioate 7-deazaguanosine H36 Cl phosphorothioate7-deazaguanosine H37 Br phosphorothioate 7-deazaguanosine H38 Iphosphorothioate 7-deazaguanosine H39 NH₂ phosphorothioate7-deazaguanosine H40 H phosphorothioate 7-deazaguanosine H41 OHphosphorodithioate 7-deazaguanosine H42 OMe phosphorodithioate7-deazaguanosine H43 F phosphorodithioate 7-deazaguanosine H44 Clphosphorodithioate 7-deazaguanosine H45 Br phosphorodithioate7-deazaguanosine H46 I phosphorodithioate 7-deazaguanosine H47 NH₂phosphorodithioate 7-deazaguanosine H48 H phosphorodithioate7-deazaguanosine H49 OH methylphosphonate 7-deazaguanosine H50 OMemethylphosphonate 7-deazaguanosine H51 F methylphosphonate7-deazaguanosine H52 Cl methylphosphonate 7-deazaguanosine H53 Brmethylphosphonate 7-deazaguanosine H54 I methylphosphonate7-deazaguanosine H55 NH₂ methylphosphonate 7-deazaguanosine H56 Hmethylphosphonate 7-deazaguanosine H57 OH boranophosphonate7-deazaguanosine H58 OMe boranophosphonate 7-deazaguanosine H59 Fboranophosphonate 7-deazaguanosine H60 Cl boranophosphonate7-deazaguanosine H61 Br boranophosphonate 7-deazaguanosine H62 Iboranophosphonate 7-deazaguanosine H63 NH₂ boranophosphonate7-deazaguanosine H64 H boranophosphonate 7-deazaguanosine I1 OH OHinosine I2 OMe OH inosine I3 F OH inosine I4 Cl OH inosine I5 Br OHinosine I6 I OH inosine I7 NH₂ OH inosine I8 H OH inosine I9 OHphosphodiester inosine I10 OMe phosphodiester inosine I11 Fphosphodiester inosine I12 Cl phosphodiester inosine I13 Brphosphodiester inosine I14 I phosphodiester inosine I15 NH₂phosphodiester inosine I16 H phosphodiester inosine I17 OHphosphonoacetate inosine I18 OMe phosphonoacetate inosine I19 Fphosphonoacetate inosine I20 Cl phosphonoacetate inosine I21 Brphosphonoacetate inosine I22 I phosphonoacetate inosine I23 NH₂phosphonoacetate inosine I24 H phosphonoacetate inosine I25 OHthiophosphonoacetate inosine I26 OMe thiophosphonoacetate inosine I27 Fthiophosphonoacetate inosine I28 Cl thiophosphonoacetate inosine I29 Brthiophosphonoacetate inosine I30 I thiophosphonoacetate inosine I31 NH₂thiophosphonoacetate inosine I32 H thiophosphonoacetate inosine I33 OHphosphorothioate inosine I34 OMe phosphorothioate inosine I35 Fphosphorothioate inosine I36 Cl phosphorothioate inosine I37 Brphosphorothioate inosine I38 I phosphorothioate inosine I39 NH₂phosphorothioate inosine I40 H phosphorothioate inosine I41 OHphosphorodithioate inosine I42 OMe phosphorodithioate inosine I43 Fphosphorodithioate inosine I44 Cl phosphorodithioate inosine I45 Brphosphorodithioate inosine I46 I phosphorodithioate inosine I47 NH₂phosphorodithioate inosine I48 H phosphorodithioate inosine I49 OHmethylphosphonate inosine I50 OMe methylphosphonate inosine I51 Fmethylphosphonate inosine I52 Cl methylphosphonate inosine I53 Brmethylphosphonate inosine I54 I methylphosphonate inosine I55 NH₂methylphosphonate inosine I56 H methylphosphonate inosine I57 OHboranophosphonate inosine I58 OMe boranophosphonate inosine I59 Fboranophosphonate inosine I60 Cl boranophosphonate inosine I61 Brboranophosphonate inosine I62 I boranophosphonate inosine I63 NH₂boranophosphonate inosine I64 H boranophosphonate inosine J1 OH OH5-methylcytidine J2 OMe OH 5-methylcytidine J3 F OH 5-methylcytidine J4Cl OH 5-methylcytidine J5 Br OH 5-methylcytidine J6 I OH5-methylcytidine J7 NH₂ OH 5-methylcytidine J8 H OH 5-methylcytidine J9OH phosphodiester 5-methylcytidine J10 OMe phosphodiester5-methylcytidine J11 F phosphodiester 5-methylcytidine J12 Clphosphodiester 5-methylcytidine J13 Br phosphodiester 5-methylcytidineJ14 I phosphodiester 5-methylcytidine J15 NH₂ phosphodiester5-methylcytidine J16 H phosphodiester 5-methylcytidine J17 OHphosphonoacetate 5-methylcytidine J18 OMe phosphonoacetate5-methylcytidine J19 F phosphonoacetate 5-methylcytidine J20 Clphosphonoacetate 5-methylcytidine J21 Br phosphonoacetate5-methylcytidine J22 I phosphonoacetate 5-methylcytidine J23 NH₂phosphonoacetate 5-methylcytidine J24 H phosphonoacetate5-methylcytidine J25 OH thiophosphonoacetate 5-methylcytidine J26 OMethiophosphonoacetate 5-methylcytidine J27 F thiophosphonoacetate5-methylcytidine J28 Cl thiophosphonoacetate 5-methylcytidine J29 Brthiophosphonoacetate 5-methylcytidine J30 I thiophosphonoacetate5-methylcytidine J31 NH₂ thiophosphonoacetate 5-methylcytidine J32 Hthiophosphonoacetate 5-methylcytidine J33 OH phosphorothioate5-methylcytidine J34 OMe phosphorothioate 5-methylcytidine J35 Fphosphorothioate 5-methylcytidine J36 Cl phosphorothioate5-methylcytidine J37 Br phosphorothioate 5-methylcytidine J38 Iphosphorothioate 5-methylcytidine J39 NH₂ phosphorothioate5-methylcytidine J40 H phosphorothioate 5-methylcytidine J41 OHphosphorodithioate 5-methylcytidine J42 OMe phosphorodithioate5-methylcytidine J43 F phosphorodithioate 5-methylcytidine J44 Clphosphorodithioate 5-methylcytidine J45 Br phosphorodithioate5-methylcytidine J46 I phosphorodithioate 5-methylcytidine J47 NH₂phosphorodithioate 5-methylcytidine J48 H phosphorodithioate5-methylcytidine J49 OH methylphosphonate 5-methylcytidine J50 OMemethylphosphonate 5-methylcytidine J51 F methylphosphonate5-methylcytidine J52 Cl methylphosphonate 5-methylcytidine J53 Brmethylphosphonate 5-methylcytidine J54 I methylphosphonate5-methylcytidine J55 NH₂ methylphosphonate 5-methylcytidine J56 Hmethylphosphonate 5-methylcytidine J57 OH boranophosphonate5-methylcytidine J58 OMe boranophosphonate 5-methylcytidine J59 Fboranophosphonate 5-methylcytidine J60 Cl boranophosphonate5-methylcytidine J61 Br boranophosphonate 5-methylcytidine J62 Iboranophosphonate 5-methylcytidine J63 NH₂ boranophosphonate5-methylcytidine J64 H boranophosphonate 5-methylcytidine K1 OH OH5-aminoallyluridine K2 OMe OH 5-aminoallyluridine K3 F OH5-aminoallyluridine K4 Cl OH 5-aminoallyluridine K5 Br OH5-aminoallyluridine K6 I OH 5-aminoallyluridine K7 NH₂ OH5-aminoallyluridine K8 H OH 5-aminoallyluridine K9 OH phosphodiester5-aminoallyluridine K10 OMe phosphodiester 5-aminoallyluridine K11 Fphosphodiester 5-aminoallyluridine K12 Cl phosphodiester5-aminoallyluridine K13 Br phosphodiester 5-aminoallyluridine K14 Iphosphodiester 5-aminoallyluridine K15 NH₂ phosphodiester5-aminoallyluridine K16 H phosphodiester 5-aminoallyluridine K17 OHphosphonoacetate 5-aminoallyluridine K18 OMe phosphonoacetate5-aminoallyluridine K19 F phosphonoacetate 5-aminoallyluridine K20 Clphosphonoacetate 5-aminoallyluridine K21 Br phosphonoacetate5-aminoallyluridine K22 I phosphonoacetate 5-aminoallyluridine K23 NH₂phosphonoacetate 5-aminoallyluridine K24 H phosphonoacetate5-aminoallyluridine K25 OH thiophosphonoacetate 5-aminoallyluridine K26OMe thiophosphonoacetate 5-aminoallyluridine K27 F thiophosphonoacetate5-aminoallyluridine K28 Cl thiophosphonoacetate 5-aminoallyluridine K29Br thiophosphonoacetate 5-aminoallyluridine K30 I thiophosphonoacetate5-aminoallyluridine K31 NH₂ thiophosphonoacetate 5-aminoallyluridine K32H thiophosphonoacetate 5-aminoallyluridine K33 OH phosphorothioate5-aminoallyluridine K34 OMe phosphorothioate 5-aminoallyluridine K35 Fphosphorothioate 5-aminoallyluridine K36 Cl phosphorothioate5-aminoallyluridine K37 Br phosphorothioate 5-aminoallyluridine K38 Iphosphorothioate 5-aminoallyluridine K39 NH₂ phosphorothioate5-aminoallyluridine K40 H phosphorothioate 5-aminoallyluridine K41 OHphosphorodithioate 5-aminoallyluridine K42 OMe phosphorodithioate5-aminoallyluridine K43 F phosphorodithioate 5-aminoallyluridine K44 Clphosphorodithioate 5-aminoallyluridine K45 Br phosphorodithioate5-aminoallyluridine K46 I phosphorodithioate 5-aminoallyluridine K47 NH₂phosphorodithioate 5-aminoallyluridine K48 H phosphorodithioate5-aminoallyluridine K49 OH methylphosphonate 5-aminoallyluridine K50 OMemethylphosphonate 5-aminoallyluridine K51 F methylphosphonate5-aminoallyluridine K52 Cl methylphosphonate 5-aminoallyluridine K53 Brmethylphosphonate 5-aminoallyluridine K54 I methylphosphonate5-aminoallyluridine K55 NH₂ methylphosphonate 5-aminoallyluridine K56 Hmethylphosphonate 5-aminoallyluridine K57 OH boranophosphonate5-aminoallyluridine K58 OMe boranophosphonate 5-aminoallyluridine K59 Fboranophosphonate 5-aminoallyluridine K60 Cl boranophosphonate5-aminoallyluridine K61 Br boranophosphonate 5-aminoallyluridine K62 Iboranophosphonate 5-aminoallyluridine K63 NH₂ boranophosphonate5-aminoallyluridine K64 H boranophosphonate 5-aminoallyluridine L1 OH OH5-methyluridine L2 OMe OH 5-methyluridine L3 F OH 5-methyluridine L4 ClOH 5-methyluridine L5 Br OH 5-methyluridine L6 I OH 5-methyluridine L7NH₂ OH 5-methyluridine L8 H OH 5-methyluridine L9 OH phosphodiester5-methyluridine L10 OMe phosphodiester 5-methyluridine L11 Fphosphodiester 5-methyluridine L12 Cl phosphodiester 5-methyluridine L13Br phosphodiester 5-methyluridine L14 I phosphodiester 5-methyluridineL15 NH₂ phosphodiester 5-methyluridine L16 H phosphodiester5-methyluridine L17 OH phosphonoacetate 5-methyluridine L18 OMephosphonoacetate 5-methyluridine L19 F phosphonoacetate 5-methyluridineL20 Cl phosphonoacetate 5-methyluridine L21 Br phosphonoacetate5-methyluridine L22 I phosphonoacetate 5-methyluridine L23 NH₂phosphonoacetate 5-methyluridine L24 H phosphonoacetate 5-methyluridineL25 OH thiophosphonoacetate 5-methyluridine L26 OMe thiophosphonoacetate5-methyluridine L27 F thiophosphonoacetate 5-methyluridine L28 Clthiophosphonoacetate 5-methyluridine L29 Br thiophosphonoacetate5-methyluridine L30 I thiophosphonoacetate 5-methyluridine L31 NH₂thiophosphonoacetate 5-methyluridine L32 H thiophosphonoacetate5-methyluridine L33 OH phosphorothioate 5-methyluridine L34 OMephosphorothioate 5-methyluridine L35 F phosphorothioate 5-methyluridineL36 Cl phosphorothioate 5-methyluridine L37 Br phosphorothioate5-methyluridine L38 I phosphorothioate 5-methyluridine L39 NH₂phosphorothioate 5-methyluridine L40 H phosphorothioate 5-methyluridineL41 OH phosphorodithioate 5-methyluridine L42 OMe phosphorodithioate5-methyluridine L43 F phosphorodithioate 5-methyluridine L44 Clphosphorodithioate 5-methyluridine L45 Br phosphorodithioate5-methyluridine L46 I phosphorodithioate 5-methyluridine L47 NH₂phosphorodithioate 5-methyluridine L48 H phosphorodithioate5-methyluridine L49 OH methylphosphonate 5-methyluridine L50 OMemethylphosphonate 5-methyluridine L51 F methylphosphonate5-methyluridine L52 Cl methylphosphonate 5-methyluridine L53 Brmethylphosphonate 5-methyluridine L54 I methylphosphonate5-methyluridine L55 NH₂ methylphosphonate 5-methyluridine L56 Hmethylphosphonate 5-methyluridine L57 OH boranophosphonate5-methyluridine L58 OMe boranophosphonate 5-methyluridine L59 Fboranophosphonate 5-methyluridine L60 Cl boranophosphonate5-methyluridine L61 Br boranophosphonate 5-methyluridine L62 Iboranophosphonate 5-methyluridine L63 NH₂ boranophosphonate5-methyluridine L64 H boranophosphonate 5-methyluridine R₁ = OH or2′modificiation R₂ = OH or internucleotide linkage B = base

As described herein, certain unnatural base pairs (e.g., isoG and isoC;Z base and P base; see Zhang et al. (2015) J. Am. Chem. Soc.) may beadvantageous for affecting the thermostability of the guide RNAsecondary structure; These modifications can be used to preventmisfolding of the guide RNA scaffold with other domains of a guide RNAsequence.

Recent guide RNA:Cas9 protein structural information (FIG. 10, asreported in Jiang et al. 2015, Science,) and in vivo/in vitro functionalmutation studies (see, e.g., Briner et al. 2014, Mol. Cell, 56, 333-9)indicate that the guide RNA scaffold is predominantly structurallyconserved. This reinforces the importance of correct folding of theconserved domain of guide RNAs for functionality with Cas9. FIG. 10shows the guide RNA scaffold secondary structure, displayinginteractions with amino acids of Cas9. Most of the guide RNA nitrogenousbases arc not involved in binding interactions with Cas9 protein.

The flanking sequences of the sgRNA scaffold increase the likelihood ofmisfolding and hence misfunction. The 20 nt guide targeting sequence, 5′of the scaffold region, is user-specified for each target, thus thelikelihood of misfolding is variable or target-specific. Also, manyemerging CRISPR-Cas applications append functional sequences 3′ of thescaffold, such as CRISPRdisplay (Schechner et al., Nat. Methods 2015)and CRISPR-i/-a (Chen et al., Cell 2013), which are riboswitches oraptamers that also need to correctly and independently fold to functionproperly. To ensure that each of the functional domains (i.e. targetingguide, scaffold, aptamer) of a given sgRNA folds in a modular,independent manner, the structurally conserved scaffold base pairs canbe substituted with unnatural, orthogonal base pairs (e.g., isoG andisoC; Z base and P base), and in some embodiments, substitutedexclusively with unnatural, orthogonal base pairs, this ensures that thesgRNA scaffold sequences will not stably interact in a secondarystructure with elements of the target-pairing guide sequence or othernon-native domains incorporated in the guide RNA such as any aptamersequences or any non-native 5′ or 3′ overhangs on the guide RNA.Alternatively, the unnatural, orthogonal base pairs mentioned abovecould be incorporated in any non-native overhangs or aptamers that maybe present, thus to prevent secondary structures involving misfolding ofthe scaffold sequence(s).

B. Guide RNA With at Least One Modification

In one aspect, the present technology provides a guide RNA having atleast one modification, constituting a modified gRNA.

In certain embodiments, the modified gRNA comprises 1, 2, 3,4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18,19, 20, 21, 22, 23, 24,25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49 or 50 modified nucleotides. In other embodiments, themodified gRNA comprises at least 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 110, 120, 130 or 140 modified nucleotides. In certain embodiments,all nucleotides are modified. In certain embodiments, all themodifications are the same. In certain embodiments, all the modifiednucleotides have the same type of modification. In certain embodiments,the modified gRNA comprises a combination of differently modifiednucleotides. In certain embodiments, the modified gRNA comprises two ormore modified nucleotides. In certain embodiments, the modified gRNAcomprises three or more modified nucleotides. In certain embodiments,the modified nucleotides arc arranged contiguously. In certainembodiments, the modified gRNA comprises at least one contiguous stretchof modified nucleotides. In certain embodiments, the modified gRNAcomprises a contiguous stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47,48, 49 or 50 modified nucleotides. Each modified nucleotide mayindependently comprise one or more types of modifications. In certainembodiments; no modified nucleotides are contiguous, or some but not allare contiguous, in the sequence of the modified gRNA.

In certain embodiments, the modification is within the 5′ portion of theguide RNA. In certain embodiments, the modification is within the firstfive (5) nucleotides of the 5′ portion of the guide RNA. In certainembodiments, the modification is within the first three (3) nucleotidesof the 5′ portion of the guide RNA. In certain embodiments, themodification is within the 3′ portion of the guide RNA. In certainembodiments, the modification is within the last five (5) nucleotides ofthe 3′ portion of the guide RNA. In certain embodiments, themodification is within the last three (3) nucleotides of the 3′ portionof the guide RNA. In certain embodiments, the:modification is within theinternal region (i.e., between the 5′ end and the 3′ end) of the guideRNA.

In certain embodiments, the modification is incorporated in the 5′portion or the 3′ portion of the guide RNA, particularly within thefirst 5 or 10 nucleotides of the 5′ portion or within the last 5 or 10nucleotides of the 3′ portion to, for example, protect the RNA fromdegradation by nucleases or for other purposes. In some otherembodiments, the modification is in both the 5′ portion and the 3′portion of the guide RNA, particularly within the first 5 or 10nucleotides of the 5′ portion and within the last 5 or 10 nucleotides ofthe 3′ portion to, for example, protect the RNA from degradation bynucleases or for other purposes. In certain embodiments, more than onetype of modification is present in both the 5′ portion and the 3′portion of the guide RNA. In certain embodiments, the modifications arelocated at the 5′ end, at the 3′ end, and within the internal sequenceof the guide RNA. In certain embodiments, a guide RNA comprises 40 orfewer, alternatively 20 or fewer, alternatively 15 or fewer,alternatively 10 or fewer, alternatively 5 or fewer, alternatively 3 orfewer deoxyribonucleotide residues in the 5′ or 3′ portion of the guideRNA.

In certain embodiments, the modification is within the crRNA segment ofthe guide RNA. In certain embodiments, the modification is within theguide sequence of the crRNA. In certain embodiments, the modification iswithin the first five (5) nucleotides of the crRNA segment. In certainembodiments, the modification is within the first three (3) nucleotidesof the crRNA segment. In certain embodiments, the modification is withina 5′-overhang on the crRNA segment. In certain embodiments, themodification is within the tracrRNA segment of the guide RNA. In certainembodiments, the modification is within the last five (5) nucleotides ofthe tracrRNA segment of the guide RNA. In certain embodiments, themodification is within the last three (3) nucleotides of the tracrRNAsegment of the guide RNA. In certain embodiments, when the guide RNA isa single guide RNA, the modification is located within the loop of theguide RNA. In certain embodiments, one or more modifications is withinthe loop L region. In certain embodiments, the modification comprises adye; a non-fluorescent label, or a tag conjugated to a linkerincorporated between two nucleotides as described above, for example byconjugation to a 2-(3-(dye/label/tag-amido)propanamido)propane-1,3-diolbis(phosphodiester) linker or to a modified base of a nucleotide m theloop or L region.

In certain embodiments, the modification comprises an end modification,such as a 5′ end modification or a 3′ end modification. Examples of endmodifications include, but are not limited to phosphorylation (asnatural phosphate or polyphosphate or as modified phosphohate groupssuch as for example, alkylphosphonate, phosphonocarboxylate,phosphonoacetate, boranophosphonate, phosphorothioate,phosphorodithioate and the like), biotinylation, conjugating orconjugated molecules, linkers, dyes, labels, tags, functional groups(such as for example but not limited to 5′-amino, 5-thio, 5-amido, 5′carboxy and the like), inverted linkages, or hydrocarbon moieties whichmay comprise ether, polyethylene glycol (PEG), ester, hydroxyl, aryl,halo, phosphodiester, bicyclic, heterocyclic or other organic functionalgroup. In certain embodiments, the end modification comprisesdimethoxytrityl.

In certain embodiments, the modification comprises a modified base. Asused herein, “unmodified” bases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Examples of modified bases include, but are not limited to,synthetic and natural bases such as 2-thioU, 2-thioG, 4-thioU, 6-thioG,2-aminoA, 2-aminoP, pseudouracil, hypoxanthine, 7-deazaguanine,7-deaza-8-azaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 5-methylC,5-methylU, 5-hydroxymethylcytosine, 5-hydroxymethyluracil,5,6-dehydrouracil, 5-propynylcytosine, 5-propynyluracil,5-ethynylcytosine, 5-ethynyluracil, 5-allylU, 5-allylC,5-aminoallyl-uracil, and 5-aminoallyl-cytosine. In certain embodiments,the modification comprises an abasic nucleotide. In certain embodiments,the modification comprises a nonstandard purine or pyrimidine structure,such as Z or P, isoC or isoG, UNA, 5-methylpyrymidinc, x(A,G,C,T) ory(A,G,C,T). In certain embodiments, the modified gRNA comprises 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 modified bases. In otherembodiments, the modified gRNA comprises at least 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 110, 120, 130 or 140 modified bases. In certainembodiments, all bases in a gRNA are modified.

In certain embodiments, the modification comprises a modified sugar.Examples of modified sugars include, but are not limited to, sugarshaving modifications, at the 21 position or modifications at the 4′position. For example, in certain embodiments, the sugar comprises2′-O-C₁₋₄alkyl, such as 2′-O-methyl (2′-OMe). In certain embodiments,the sugar comprises 2′-O-C₁₋₃alkyl-O-C₁₋₃alkyl, such as 2′-methoxyethoxy(2′-O-CH₂CH₂OCH₃) also known as 2′-O-(2-methoxyethyl) or 2′-MOE. Incertain embodiments, the sugar comprises 2′-halo, such as 2′-F, 2′-Br,2′-Cl, or 2′-I. In certain embodiments, the sugar comprises 2′-NH₂. Incertain embodiments; the sugar comprises 2′-H (e.g., a deoxynucleotide).In certain embodiments, the sugar comprises 2′-arabino or 2′-F-arabino.In certain embodiments, the sugar comprises 2′-LNA or 2′-ULNA. Incertain embodiments, the sugar comprises a 4′-thioribosyl. In certainembodiments, the modified gRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49 or 50 modified sugars. In other embodiments, the modifiedgRNA comprises at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110,120, 130 or 140 modified sugars. In certain embodiments, all sugars in agRNA are modified.

In certain embodiments, the modification comprises a modified backbone(i.e., an internucleotide linkage other than a natural phosphodiester).Examples of modified internucleotide linkages include, but are notlimited to a phosphorothioate internucleotide linkage, a chiralphosphorothioate internucleotide linkage, a phosphorodithioateinternucleotide linkage, a boranophosphonate internucleotide linkage, aC₁₋₄alkyl phosphonate internucleotide linkage such as amethylphosphonate internucleotide linkage, a boranophosphonateinternucleotide linkage, a phosphonocarboxylate internucleotide linkagesuch as a phosphonoacetate internucleotide linkage, aphosphonocarboxylate ester internucleotide linkage such as aphosphonoacetate ester internucleotide linkage, athiophosphonocarboxylate internucleotide linkage such as for example athiophosphonoacetate internucleotide linkage, a thiophosphonocarboxylateester internucleotide linkage such as a thiophosphonoacetate esterinternucleotide linkage. Various salts, mixed salts and free acid formsare also included. In certain embodiments, the modified gRNA comprises1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 modifiedinternucleotide linkages. In other embodiments, the modified gRNAcomprises at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120,130 or 140 modified internucleotide linkages. In certain embodiments,all internucleotide linkages in a gRNA are modified.

In certain embodiments, the modification is a 2′-O-C₁₋₃alkyl, 2′-H,2′-O-C₁₋₃alkyl-O-C₁₋₃alkyl, 2-F, 2′-NH₂, 2′-arabino, 2′-F-arabino,2′-LNA, 2′-ULNA, 4′-thioribosyl, 2-thioU, 2-thioC, 4-thioU, 6-thioG,2-aminoA, 2-aminoP, pseudouracil, hypoxanthine, 7-deazaguariine,7-deaza-8-azaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 5-MeC,5-MeU, 5-hydroxymethylcytosine, 5-hydroxymethyluracil,5,6-dehydrouracil, 5-propynylcytosine, 5-propynyluracil,5-ethynylcytosine, 5-ethynyluracil, 5-allylU, 5-allylC,5-aminoallyl-uracil, 5-aminoallyl-cytosine, an abasic nucleotide, Z, P,UNA, isoC, isoG, 5-methyl-pyrimidine, x(A,G,C,T), y(A,G,C,T), a3-phosphorothioate group, a 3′-phosphonoacetate group, a3′-phosphonoacetate ester-group, a 3′-thiophosphonoacetate group, a3′-thiophosphonoacetate ester group, a 3′-methylphosphonate group, a3-boranophosphonate group, a 3′-phosphorodithioate group, orcombinations thereof.

In certain embodiments, the modified nucleotide comprises a2′-O-methyl-3-phosphorothioate. In certain embodiments, the modifiednucleotide comprises a 2′-O-methyl-3′-phosphonoacetate. In certainembodiments, the modified nucleotide comprises a2′-O-methyl-3′-thiophosphonoacetate. In certain embodiments, themodified nucleotide comprises a Z base. In certain embodiments, themodified nucleotide comprises a 2′-halo-3′-phosphorothioate. In certainembodiments, the modified nucleotide comprises a2′-halo-3-phosphonoacetate. In certain embodiments, the modifiednucleotide comprises a 2′-halo-3′-thiophosphonoacetate. In certainembodiments, the modified nucleotide comprises a2′-fluoro-3′-phosphorothioate. In certain embodiments, the modifiednucleotide comprises a 2′-fluoro-3′-phosphonoacetate. In certainembodiments, the modified nucleotide comprises a2′-fluoro-3′-thiophosphonoacetate.

In certain embodiments, the guide RNA comprises an oligonucleotiderepresented by Formula (I):

W-Y or Y-W   (I)

wherein W represents a nucleotide or a stretch of nucleotides of theoligonucleotide comprising at least one modification and Y represents anunmodified portion of the oligonucleotide.

In certain embodiments, W is within the 5′ portion of the guide RNA. Incertain embodiments, W is at least partially within the first five (5)nucleotides of the 5′ portion of the guide RNA. In certain embodiments,W is at least partially within the first three (3) nucleotides of the 5′portion of the guide RNA. In certain embodiments, W is within the 3′portion of the guide RNA. In certain embodiments, W is at leastpartially within the last five (5) nucleotides of the 3′ portion of theguide RNA. In certain embodiments, W is at least partially within thelast three (3) nucleotides of the 3′ portion of the guide RNA. Incertain embodiments, W is within the internal region (i.e., between the5′ end and the 3′ end) of the guide RNA.

In certain embodiments, W comprises an end modification, such as a 5′end modification or a 3′ end modification as described above. In certainembodiments, the end modification comprises dimethoxytrityl.

In certain embodiments, W comprises a modified base as described above.In certain embodiments, W comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49 or 50 modified bases. In other embodiments, W comprises at least55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130 or 140 modifiedbases. In certain embodiments, all bases in a gRNA are modified.

In certain embodiments, W comprises a modified sugar as described above.In certain embodiments, W comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49 or 50 modified sugars. In other embodiments, W comprises at least55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130 or 140 modifiedsugars. In certain embodiments, all sugars in a gRNA are modified.

In certain embodiments, W comprises a modified backbone (i.e., aninternucleotide linkage other than a phosphodiester) as described above.In certain embodiments; W comprises more than one, such as 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49 or 50 modified internucleotide linkages. Inother embodiments, W comprises at least 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 110, 120, 130 or 140 modified internucleotide linkages. Incertain embodiments, all internucleotide linkages in a gRNA aremodified.

In certain embodiments, W comprises a 2′-O-C₁₋₄alkyl, 2′-H,2′-O-C₁₋₃alkyl-O-C₁₋₃alkyl, 2′-F, 2′-NH₂, 2′-arabino, 2′-F-arabino,2′-LNA, 2′-ULNA, 4′-thioribosyl, 2-thioU, 2-thioC, 4-thioU, 6-thioG,2-aminoA, 2-aminoP, pseudouracil, hypoxanthine; 7-deazaguanine,7-deaza-8-azaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 5-MeC,5-MeU, 5-hydroxymethylcytosine, 5-hydroxymethyluracil,5,6-dehydrouracil, 5-propynylcytosine, 5-propynyluracil,5-ethynylcytosine, 5-ethynyluracil, 5-allylU, 5-allylC,5-aminoallyl-uracil, 5-aminoallyl-cytosine, abasic nucleotides, Z, P,UNA, isoC, isoG, 5-methyl-pyrimidine, x(A,G,C,T), y(A,G,C,T), aphosphorothioate internucleotide linkage, a phosphonoacetateinternucleotide linkage, a phosphonoacetate ester internucleotidelinkage, a thiophosphonoacetate internucleotide linkage, athiophosphonoacetate ester internucleotide linkage a methylphosphonateinternucleotide linkage, a boranophosphonate internucleotide linkage, aphosphorodithioate internucleotide linkage, or combinations thereof.

In certain embodiments, W comprises a 2′-O-methyl and a3′-phosphorothioate group on the same nucleotide. In certainembodiments, W comprises a 2′-O-methyl and a 3′-phosphonoacetate groupon the same nucleotide. In certain embodiments, W comprises a2′-O-methyl and 3′-thiophosphonoacetate group on the same nucleotide. Incertain embodiments, W comprises a 2′-F and a 3′-phosphorothioate groupon the same nucleotide. In certain embodiments, W comprises 2′-F and a3′-phosphonoacetate group on the same nucleotide. In certainembodiments, W comprises a 2′-F and 3′-thiophosphonoacetate group on thesame nucleotide.

In certain embodiments, W comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19 or 20 modified nucleotides. In certainembodimerits, each of the modified nucleotides comprises the samemodification. In certain embodimerits, W comprises a combination ofvariously modified nucleotides. In certain embodiments, W comprises twoor more modified nucleotides. In certain embodiments, W comprises threeor more modified nucleotides. In certain embodiments, the modifiednucleotides are not arranged contiguously in the sequence, or at leastnot entirely, as one or more unmodified nucleotides may intercede. Incertain embodiments, the modified nucleotides are arranged contiguously.In certain embodiments, W comprises at least one contiguous stretch ofmodified nucleotides. In certain embodiments, W comprises a contiguousstretch of at least three (3) modified nucleotides. In certainembodiments, W comprises a contiguous stretch of at least four (4)modified nucleotides. In certain embodiments, W comprises a contiguousstretch of at least five (5) modified nucleotides.

In certain embodiments, the guide RNA comprises an oligonucleotiderepresented by Formula (II):

M_(m)N_(n)   (II)

wherein each N independently represents an unmodified ribonucleotide;

wherein each M represents a modified nucleotide and is independentlyselected from the group consisting of a 2′-O-methyl ribonucleotide, a3′-P(S) ribonucleotide, a 3′-PACE ribonucleotide, a 3′-thioPACEribonucleotide, a 2′-O-methyl-3′-P(S)-ribonucleotide, a2′-O-methyl-3′-PACE ribonucleotide, a 2′-O-methyl-3′-thioPACEribonucleotide, a Z nucleotide, and a 2′-deoxynucleotide;

wherein each M is at any position of the sequence of the guide RNA;

wherein any given M is the same or different from any other M, and anygiven N is the same or different from any other N; and

wherein each of m and n are independently selected from an integerbetween 0 and 219, provided that 50<m+n≦220, and m is not 0.

In some embodiments, m+n<150.

In certain embodiments, each M is modified with one or more moietiesindependently selected from the group consisting of 2-F, 2-thiouracil,4-thiouracil, 2-aminoadenine, hypoxanthine, 5-methylcytosine,5-methyluracil, 5-allylaminouracil, squarate linkage, a triazololinkage, and a 2-(4-butylamidofluorescein)propane-1,3-diolbis(phosphodiester) linkage. In some embodiments, M comprises a dyeattached through a linker.

In certain embodiments, each M is independently selected from the groupconsisting of a 2′-O-methyl ribonucleotide, a 2′-O-methyl-3′-P(S)ribonucleotide, a 2′-O-methyl-3′-PACE ribonucleotide, and a2′-O-methyl-3′-thioPACE ribonucleotide. In certain embodiments, each Mis independently selected from the group consisting of a2′-O-methyl-3′-pACE ribonucleotide and a 2′-O-methyl-3′-thioPACEribonucleotide.

In certain embodiments, where m>1, any given M is the same or differentfrom any other M. In certain embodiments, where m>1, each M has the samemodification.

In certain embodiments, each M is a 2′-O-methyl-3′-PACE ribonucleotide,m is selected from an integer between 1 and 10, each N is independentlyselected from the group consisting of A, U, C, and G, and n is selectedfrom an integer between 1 and 149, provided 50<m+n≦150. In certainembodiments, each M is a 2′-O-methyl-3′-PACE ribonucleotide, m isselected from an integer between 1 and 5, each N is independentlyselected from the group consisting of A, U, C, and G, and n is selectedfrom an integer between 1 and 149, provided 50<m+n≦150. In certainembodiments, each M is a 2′-O-methyl-3′-PACE ribonucleotide, m isselected from an integer between 2 and 5, each N is independentlyselected from the group consisting of A, U, C, and G, and n is selectedfrom an integer between 1 and 148, provided 50<m+n≦150. In certainembodiments, m is 1. In certain embodiments, m is 2. In certainembodiments, m is 3. In certain embodiments, m is 4. In certainembodiments, m is 5.

In certain embodiments, each M is a 2′-O-methyl-3′-thioPACEribonucleotide, m is selected from an integer between 1 and 10, each Nis independently selected from the group consisting of A, U, C, and G,and n is selected from an integer between 1 and 149, provided50<m+n≦150. In certain embodiments, each M is a 2′-O-methyl-3′-thioPACEribonucleotide, m is selected from an integer between 1 and 5, each N isindependently selected from the group consisting of A, U, C, and G, andn is selected from an integer between 1 and 149, provided 50<m+n≦150. Incertain embodiments, each M is a 2′-O-methyl-3′-thioPACE ribonucleotide,m is selected from an integer between 2 and 5, each N is independentlyselected from the group consisting of A, U, C, and G, and n is selectedfrom an integer between 1 and 148, provided 50<m+n≦150. In certainembodiments, m is 1. In certain embodiments, m is 2. In certainembodiments, m is 3. In certain embodiments, m is 4. In certainembodiments, m is 5.

In certain embodiments, each M is a 2′-O-methyl ribonucleotide, m isselected from an integer between 1 and 40, each N is independentlyselected from the group consisting of A, U, C, and G, and n is selectedfrom an integer between 1 and 149, provided 50<m+n≦150. In certainembodiments, each M is a 2′-O-methyl ribonucleotide, m is selected froman integer between 1 and 25, each N is independently selected from thegroup consisting of A, U, C, and G, and n is selected from an integerbetween 1 and 149; provided 50<m+n≦150. In certain embodiments, each Mis a 2′-O-methyl ribonucleotide, m is selected from an integer between 1and 20, each N is independently selected from the group consisting of A,U, C, and G, and n is selected from an integer between 1 and 149,provided 50<m+n≦150. In certain embodiments, m is 1. In certainembodiments, m is 2. In certain embodiments, m is 3. In certainembodiments, m is 4. In certain embodiments, m is 5. In certainembodiments, m is 10. In certain embodiments, m is 15. In certainembodiments, m is 20. In certain embodiments, m is 30. In certainembodiments, m is 40.

In certain embodiments, each M is a 2′-deoxynucleotide, m is selectedfrom an integer between 1 and 30, each N is independently selected fromthe group consisting of A, U, C, and G, and n is selected from aninteger between 1 and 149, provided 50<m+n≦150. In certain embodiments,each M is 2′-deoxynucleotide, m is selected from an integer between 1and 20, each N is independently selected from the group consisting of A,U, C, and G, and n is selected from an integer between 1 and 149,provided 50<m+n≦150. In certain embodiments, m is 5. In certainembodiments, m is 10. In certain embodiments, m is 15. In certainembodiments, m is 20. In certain embodiments, m is 30.

In certain embodiments, each M is a 2′-O-methyl-3′-P(S) ribonucleotide,m is selected from an integer between 1 and 10, each N is independentlyselected from the, group consisting of A, U, C, and G, and n is selectedfrom an integer between 1 and 149, provided 50<m+n≦150. In certainembodiments, each M is a 2′-O-methyl-3′-P(S) ribonucleotide, m isselected from an integer between 1 and 5, each N is independentlyselected from the group consisting of A, U, C, and G, and n is selectedfrom an integer between 1 and 149, provided 50<m+n≦150. In certainembodiments, m is 1. In certain embodiments, m is 2. In certainembodiments, m is 3. In certain embodiments, m is 4. In certainembodiments, m is 5.

In certain embodiments, each M is a Z nucleotide, m is selected from aninteger between 1 and 10, each N is independently selected from thegroup consisting of A, U, C, and G, and n is selected from an integerbetween 1 and 149, provided 50<m+n≦150. In certain embodiments, each Mis a Z nucleotide, m is selected from an integer between 1 and 5, each Nis independently selected from the group consisting of A, U, C, and G,and n is selected from an integer between 1 and 149, provided50<m+n≦150. In certain embodiments, m is 1. In certain embodiments, m is2. In certain embodiments, m is 3. In certain embodiments, m is 4. Incertain embodiments, m is 5.

In certain embodiments, the modification is a stability-alteringmodification. In certain embodiments, the modification increasesnuclease resistance of the guide RNA relative to a guide RNA without themodification, thus it enhances the guide RNA stability. In certainembodiments, the stability-altering modification is astability-enhancing modification. For example, in certain embodiments,the stability-enhancing modification comprises, a 2′-O-methyl or a2′-O-C₁₋₄alkyl nucleotide. In certain embodiments, thestability-enhancing modification comprises a 2-halo nucleotide, such as2′-F, 2′-Br, 2′-Cl, or 2′-I. In certain embodiments, thestability-enhancing modification comprises a 2′MOE or a2′-O-C₁₋₃alkyl-O-C₁₋₃alkyl. In certain embodiments, thestability-enhancing modification comprises a 2′-NH₂ nucleotide. Incertain embodiments, the stability-enhancing modification comprises a2′-H (or 2′-deoxy) nucleotide. In certain embodiments, thestability-enhancing modification comprises a 2′-arabino or a2′-F-arabino. In certain embodiments, the stability-enhancingmodification comprises a 4′-thioribosyl sugar moiety. In certainembodiments, the stability-enhancing modification comprises a3′-phosphorothioate group. In certain embodiments, thestability-enhancing modification comprises a 3′-phosphonoacetate group.In certain embodiments, the stability-enhancing modification comprises anucleotide containing a 3′-thiophosphonoacetate group. In certainembodiments, the stability-enhancing modification comprises a nucleotidecontaining a 3′-methylphosphonate group. In certain embodiments, thestability-enhancing modification comprises a nucleotide containing a3′-boranophosphate group. In certain embodiments, thestability-enhancing modification -comprises a nucleotide containing a3′-phosphorodithioate group. In certain embodiments, thestability-enhancing modification comprises a locked nucleic acid (“LNA”)nucleotide. In certain, embodiments, the stability-enhancingmodification comprises an unlocked nucleic acid (“ULNA”) nucleotide.

In certain embodiments, the stability-enhancing modification comprises a2′-O-methyl and a 3′-phosphorothioate group on the same nucleotide. Incertain embodiments, the stability-enhancing modification comprises a2′-O-methyl and a 3′-phosphonoacetate group on the same nucleotide. Incertain embodiments, the stability-enhancing modification comprises a2′-O-methyl and a 3′-thiophosphonoacetate group on the same nucleotide.In certain embodiments, the stability-enhancing modification comprises a2′-fluoro and a 3′-phosphorothioate group on the same nucleotide. Incertain embodiments, the stability-enhancing modification comprises a2′-fluoro and a 3′-phosphonoacetate group on the same nucleotide. Incertain embodiments, the stability-enhancing modification comprises a2′-fluoro and a 3′-thiophosphonoacetate group on the same nucleotide.

In certain embodiments, the modification is a specificity-alteringmodification. In some embodiments, specificity enhancement may beachieved by enhancing on-target binding and/or cleavage, or reducingoff-target binding and/or cleavage, or a combination of both. In someother embodiments, specificity reduction may be achieved, for example,by reducing on-target binding and/or cleavage, or increasing off-targetbinding and/or cleavage, or a combination of both.

In certain embodiments, the specificity-altering modification comprisesa 2′-O-methyl. In certain embodiments the specificity-alteringmodification comprises a 2′-halo, such as 2′-fluoro.

In certain embodiments, the specificity-altering modification comprisesa 2-thiouracil base (2-thioU). In certain embodiments, thespecificity-altering modification comprises 2-thioC. In certainembodiments, the specificity-altering modification comprises 4-thioU. Incertain embodiments, the specificity-altering modification comprises6-thioG. In certain embodiments, the specificity-altering modificationcomprises 2-aminoA. In certain embodiments, the specificity-alteringmodification comprises a 2-aminopurine. In certain embodiments, thespecificity-altering modification comprises pseudouracil. In certainembodiments, the specificity-altering modification compriseshypoxanthine. In certain embodiments, the specificity-alteringmodification comprises 7-deazaguanine. In certain embodiments, thespecificity-altering modification comprises 7-deaza-8-azaguanine. Incertain embodiments, the specificity-altering modification comprises7-deazaadenine. In certain embodiments, the specificity-alteringmodification comprises 7-deaza-8-azaadenine. In certain embodiments, thespecificity-altering modification comprises 5-methylC. In certainembodiments, the specificity-altering modification comprises 5-methylU.In certain embodiments, the specificity-altering modification comprises5-hydroxymethylcytosine. In certain embodiments, thespecificity-altering modification comprises 5-hydroxymethyluracil. Incertain embodiments, the specificity-altering modification, comprises5,6-dehydrouracil. In certain embodiments, the specificity alteringmodification comprises 5-propynylcytosine. In certain embodiments, thespecificity-altering modification comprises 5-propynyluracil. In certainembodiments, the specificity-altering modification comprises5-ethynylcytosine. In certain embodiments, the specificity-alteringmodification comprises 5-ethynyluracil. In certain embodiments, thespecificity-altering modification comprises 5-allylU. In certainembodiments, the specificity-altering modification comprises 5-allylC.In certain embodiments, the specificity-altering modification comprises5-aminoallylU. In certain embodiments, the specificity-alteringmodification comprises 5-aminoallylC. In certain embodiments, thespecificity-altering modification comprises an abasic nucleotide. Incertain embodiments, the specificity-altering modification comprises a Zbase. In certain embodiments, the specificity-altering modificationcomprises P base. In certain embodiments, the specificity-alteringmodification comprises a UNA base. In certain embodiments, thespecificity-altering modification comprises isoC. In certainembodiments, the specificity-altering modification comprises isoG. Incertain embodiments, the specificity-altering modification comprises5-methyl-pyrimidine. In certain embodiments, the specificity-alteringmodification comprises x(A,G,C,T). In certain embodiments, thespecificity-altering modification comprises y(A,G,C,T).

In certain embodiments, the specificity-altering modification comprisesa phosphorothioate internucleotide linkage. In certain embodiments, thespecificity-altering modification comprises a phosphonoacetateinternucleotide linkage. In certain embodiments, thespecificity-altering modification comprises a thiophosphonoacetateinternucleotide linkage. In certain embodiments, thespecificity-altering modification comprises a methylphosphonateinternucleotide linkage. In certain embodiments, thespecificity-altering modification comprises a boranophosphateinternucleotide linkage. In certain embodiments, thespecificity-altering modification comprises a phosphorodithioateinternucleotide linkage. In certain embodiments, thespecificity-altering modification comprises a ULNA. In certainembodiments, the specificity-altering modification comprises an LNA.

In certain embodiments, the modification alters RNA base pairing by, forexample, altering the melting temperature (Tm) of the guide RNA relativeto a guide RNA without the modification. In certain embodiments, themodification lowers the Tm of the guide RNA relative to a guide RNAwithout the modification. In certain embodiments, the modificationraises the Tm of the guide RNA relative to a guide RNA without themodification.

In certain embodiments, the specificity-altering modification lowers theTm of a base pairing interaction. In certain embodiments, themodification that lowers the Tm of the base pairing interaction is a2′-deoxy, as it is well-known in the art that DNA/DNA base pairs havelower Tm than their respective counterpart in RNA/DNA duplexes. Incertain embodiments, the modification that lowers the Tm of the basepairing interaction is 2-thiouracil, which slightly lowers Tm of G-Uwobble pair. In certain embodiments, the modification that lowers the Tmof the base pairing interaction is a phosphorothioate internucleotidelinkage or a phosphorodithioate internucleotide linkage, which lower theTm by ˜0.5° C. per modification. In certain embodiments, themodification that lowers the Tm of the base pairing, interaction is aboranophosphonate internucleotide linkage, which lowers the Tm by˜0.5-0.8° C. per modification. In certain embodiments, the modificationthat lowers the Tm of the base pairing interaction is a phosphonoacetateinternucleotide linkage, which lowers the Tm by ˜1.3° C. permodification. In certain embodiments, the modification that lowers theTm of the base pairing interaction is unlocked nucleic acid (“ULNA”),which lowers the Tm by ˜5-8° C. per modification. In certainembodiments, the modification that lowers the Tm of the base pairinginteraction is 2-O-methyl-3-methylphosphonate.

In certain embodiments, the specificity-altering modification raises theTm of a base pairing interaction. In certain embodiments, themodification that raises the Tm of the base pairing interaction is a2′-O-methyl, which raises Tm by ˜0.5-0.7° C. per modification. Incertain embodiments, the modification that raises the Tm of the basepairing interaction is a 2′-F, which raises Tm by ˜1° C. permodification. In certain embodiments, the modification that raises theTm of the base pairing interaction is a 2-thiouracil, which raises Tm ofA-U pair (and, as noted above, slightly lowers Tm of G-U wobble pair).In certain embodiments, the modification that raises the tm of the basepairing interaction is a 4-thiouracil, which raises Tm of G-U wobblepair and slightly raises Tm of A-U pair. In certain embodiments, themodification that raises the Tm of the base pairing interaction is a2-amino-adenine, which raises tm of its base pairing with U by ˜1° C.per modification. In certain embodiments, the modification that raisesthe Tm of the base pairing interaction is a 5-methyl-uracil (5-methylU)(see, e.g., Wang & Kool (1995) Biochemistry, 34, 4125-32). In certainembodiments, the modification that raises the Tm of the base pairinginteraction is a 5-methyl-cytosine (5-methylC). In certain embodiments,the modification that raises the Tm of the base pairing interaction is alocked nucleic acid (“LNA”), which raises Tm by 2-10° C. permodification.

In certain embodiments/the modification alters transfection efficiencyof the guide RNA relative to a guide RNA without the modification. Incertain embodiments, the modification increases transfection efficiencyof the guide RNA relative to a guide RNA without the modification: Incertain embodiments, the modification decreases transfection efficiencyof the guide RNA relative to a guide RNA without the modification. Incertain embodiments, the modification neutralizes the anionic charge onphosphate to allow passive diffusion into cells. In certain embodiments,the charge-neutralizing modification comprises a phosphonoacetate alkylester internucleotide linkage, such as a phosphonoacetate methyl esterinternucleotide linkage.

In certain embodiments, the modification alters the immunostimulatoryeffect of the guide RNA relative to a guide RNA without themodification. It was initially discovered that unmethylated bacterialDNA and synthetic analogs thereof are ligands for TLR9 (see Hemmi et al.(2000) Nature, 408, 740-5). The stimulation of TLR9 can be mitigated inthe dinucleotide motif for example by modifying the C and G -residues.The use of 5-methylcytosine, 2-aminocytosine, 2-thiocytosine,5-methylisocytosine, P nucleobase;(6-(β-D-2′-Deoxyribofuranosyl)-3,4-dihydro-8H-pyrimido[4,5-c][1,2]oxazin-7-one),and 2′-O-methylcytosine all result in loss or decrease in TLR9stimulation. In certain embodiments, use of 6-thioguanine,2,6-diaminopurine, 2-aminopurine, xanthosine, inosine,7-deazaxanthosine, isoguanine, 8-oxoguanine, nebularine, 8-bromoguanine,K-nucleobase (2-amino-N-methoxyadenosine), and/or 2′-O-methylguanine canresult in loss or decrease in TLR9 stimulation. In some embodiments, useof phosphodiester modifications can lower or eliminate the TLR9response. Typically, synthetically incorporated phosphorothioates candecrease the TLR9 response to a limited extent, as is thought to resultfrom the presence of two stereoisomers of each phosphorothioate insynthetic RNA. However, it has been shown that phosphorothioate-modifiedDNA lacking CpG motifs stimulate TLR9 to a rather small extent. Thenegative charge on the phosphorus is an important element forrecognition by TLR9 and therefore removing the negative charge usingalkylphosphonate can result in loss or decrease in TLR9 stimulation. Theuse of phosphonoacetate (PAGE) internucleotide linkages betweendeoxynucleotides in 5′ and 3′ terminal sequences can significantlyincrease the TLR9 response; however, the use of thiophosphonoacetate(thioPACE) internucleotide linkages between deoxynucleotides in 5′ and3′ terminal sequences can result in loss or decrease in TLR9stimulation. In certain embodiments, use of sugar modifications that thefavor C3′ -endo conformation such as 2′-O-methyl modifications can beincorporated at 5′ and 3′ termini to decrease the TLR9 response. TLR 7and TLR8 can be stimulated by molecules containing 7-deazaguanine and bysingle-stranded RNA (see e.g., Heil et al. (2004) Science, 303, 1526-9).TLR3 has been implicated in cellular immunoresponses to virus-deriveddouble-stranded RNA. In certain embodiments, these TLR responses can bemitigated for example by using 2′-O-methyl modifications, modifiedphosphodiester linkages containing sulfur, or modifications thatdecrease internucleotide negative charge such as methylphosphonateand/or phosphonoacetate internucleotide linkages.

In certain embodiments, the modification enhances stability andspecificity of the guide RNA relative to a guide RNA without themodification. In certain embodiments, the modification enhancesstability and transfection efficiency of the guide RNA relative to aguide-RNA without the modification. In certain embodiments, themodification enhances specificity and transfection efficiency of theguide RNA relative to a guide RNA without the modification. In certainembodiments, the modification enhances the overall efficacy of the guideRNA relative to a guide RNA without the modification.

C. Guide RNA with a Combination of Modifications

In one aspect, the present technology provides a guide RNA having acombination of two or more modifications.

In certain embodiments, the two modifications are on the same nucleotide(for example, one nucleotide comprises a 2′-O-methyl and a3′-thiophosphonoacetate moiety). In other embodiments, the twomodifications are on two different nucleotides (for example, onenucleotide has a 2-thioU base and another nucleotide has a 2-O-methylgroup).

In certain embodiments, each modification in the guide RNA is the same.In certain embodiments, at least one modification in the guide RNA isdifferent from at least one other modification in the guide RNA. Incertain embodiments, a single nucleotide within the guide RNA possessestwo or more modifications.

In certain embodiments, the guide RNA comprises a combination ofdifferent types of modifications, and at least one type in thecohibination exists in multiple places in the guide RNA. In certainembodiments, at least one type in the combination appears 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 times in theguide RNA.

In certain embodimerits, at least one type of the modifications in thecombination appears in two or more modified nucleotides. In certainembodiments, at least one type of the modifications in the combinationappears in three or more modified nucleotides. In certain embodiments,the modified nucleotides are not arranged contiguously in the sequence,or at least not entirely, as one or more unmodified nucleotides mayintercede. In certain embodiments, the modified nucleotides are arrangedcontiguously. In certain embodiments, the guide RNA comprises a stretchof contiguous modified nucleotides of the same type. In certainembodiments, the stretch has at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 modified nucleotides.

In certain embodiments, at least one type of the modifications in thecombination is within the 5′ portion of the guide RNA. In certainembodiments, at least one type of the modifications in the combinationis within the first five (5) nucleotides of the 5′ portion of the guideRNA. In certain embodiments, at least one type of the modifications inthe combination is within the first three (3) nucleotides of the 5′portion of the guide RNA. In certain embodiments, at least one type ofthe modifications in the combination is within the 3′ portion of theguide RNA. In certain embodiments, at least one type of themodifications in the combination is within the last five (5) nucleotidesof the 3′ portion of the guide RNA. In certain embodiments, at least onetype of the modifications in the combination is within the last three(3) nucleotides of the 3′ portion of the guide RNA. In certainembodiments, at least one type of the modifications in the conciliationis within the internal region (i.e., between the 5′ end and the 3′ end)of the guide RNA.

In certain embodiments, at least one type of the modifications in thecombination is incorporated in the 5′ portion or 3′ portion of the guideRNA, particularly within the first 5 or 10 nucleotides of the 5′ portionor within the last 5 or 10 nucleotides of the 3′ portion to, forexample, protect the RNA from degradation by nucleases or for otherpurposes. In certain embodiments, at least one type of the modificationsin the combination is in the 5′ portion and at least one type of themodifications in the combination is in the 3′ portion of the guide RNA,particularly within the first 5 or 10 nucleotides of the 5′ portion andwithin the last 5 or 10 nucleotides of the 3′ portion to, for example,protect the RNA from degradation by nucleases or for other purposes. Incertain embodiments, a guide RNA comprises 20 or fewer, alternatively 15or fewer, alternatively 15 or fewer, alternatively 10 or fewer,alternatively 5 or fewer, alternatively 3 or fewer deoxyribonucleotideresidues in the 5′ portion of the guide RNA.

In certain embodiments, at least one type of the modifications in thecohibination is within the crRNA segment of the guide RNA. In certainembodiments, at least one type of the modifications in the combinationis within the guide sequence of the crRNA. In certain embodiments, atleast One type of the modifications in the combination is within thefirst five (5) nucleotides of the crRNA segment. In certain embodiments,at least one type of the modifications in the combination is within, thefirst three (3) nucleotides of the crRNA segment. In certainembodiments, at least one type of the modifications in the combinationis within the tracrRNA segment of the guide RNA. In certain embodiments,at least one type of the modifications in the combination is within thelast five (5) nucleotides of the tracrRNA segment of the guide RNA. Incertain embodiments, at least one type of the modifications in thecombination is within the last three (3) nucleotides of the tracrRNAsegment of the guide RNA.

In certain embodiments, a first type of modification in thecombination's within the 5′ portion of the guide RNA and a second typeof modification in the combination is within the internal region (i.e.,between the 5′ end and the 3′ end) of the guide RNA. In certainembodiments, the first type of modification is within the first five (5)nucleotides of the 5′ portion of the guide RNA. In certain embodiments,the first type of modification is within the first three (3) nucleotidesof the 5′ portion of the guide RNA.

In certain embodiments, a first type of modification in the combinationis within the internal region (i.e., between the 5′ end and the 3′ end)of the guide RNA and a second type of modification in the combination iswithin the 3′ portion of the guide RNA. In certain embodiments, thesecond type of modification is within the last five (5) nucleotides ofthe 3′ portion of the guide RNA. In certain embodiments; the second typeof modification is within the last three (3) nucleotides of the 3′portion of the guide RNA.

In certain embodiments, a first type of modification in the combinationis within the 5′ portion of the guide RNA and a second type ofmodification in the combination is within the 3′ portion of the guideRNA. In certain embodiments, the first type of modification is withinthe first five (5) nucleotides of the 5′ portion of the guide RNA. Incertain embodiments, the first type of modification is within the firstthree (3) nucleotides of the 5′ portion of the guide RNA. In certainembodiments, the second type of modification is within the last five (5)nucleotides of the 3′ portion of the guide RNA. In certain embodiments,the second type of modification is within the last three (3) nucleotidesof the 3′ portion of the guide RNA.

In certain embodiments, a first type of modification in the combinationis within the 5′ portion of the guide RNA, a second type of modificationin the combination is within the internal region (i.e., between the 5′end and the 3′ end) of the guide RNA, and a third type of modificationin the combination is within the 3′ portion of the guide RNA. In certainembodiments, the first type of modification is within the first five (5)nucleotides of the 5′ portion of the guide RNA. In certain embodiments,the first type of modification is within the first three (3) nucleotidesof the 5′ portion of the guide RNA. In certain embodiments, the thirdtype of modification is within the last five (5) nucleotides of the 3′portion of the guide RNA. In certain embodiments, the third type ofmodification is within the last three (3) nucleotides of the 3′ portionof the guide RNA.

In certain embodiments, a first type of modification in the combinationis within the crRNA segment of the guide RNA and a second type ofmodification in the combination is within the trace segment. In certainembodiments, the first type of modification is within the guide sequenceof the crRNA. In certain embodiments, the first type of modification iswithin the first five (5) nucleotides of the crRNA segment. In certainembodiments, the first type of modification is within the first three(3) nucleotides of the crRNA segment. In certain embodiments, the secondtype of modification is within the last five (5) nucleotides of thetracrRNA segment of the guide RNA. In certain embodiments, the secondtype of modification is within the last three (3) nucleotides of thetracrRNA segment of the guide RNA.

In certain embodiments, a first type and a second type of modificationin the combination are within the crRNA segment of the guide RNA. Incertain embodiments, the first type of modification is within the guidesequence of the crRNA. In certain embodiments, the first type ofmodification is Within the first five (5) nucleotides of the crRNAsegment. In certain embodiments, the first type of modification iswithin the first three (3) nucleotides of the crRNA segment.

In certain embodiments, a first type and a second type of modificationin the combination are within the crRNA segment of the guide RNA and athird type of modification in the combination is within the tracesegment. In certain embodiments, the first type of modification iswithin the guide sequence of the crRNA. In certain embodiments, thefirst type of modification is within the first five (5) nucleotides ofthe crRNA segment. In certain embodiments, the first type-ofmodification is within the first three (3) nucleotides of the crRNAsegment. In certain embodiments, the third type of modification iswithin the last five (5) nucleotides of the tracrRNA segment of theguide RNA. In certain embodiments, the third type of modification iswithin the last three (3) nucleotides of the tracrRNA segment of theguide RNA.

In certain embodiments, at least one of the modifications in thecombination comprises an end modification, such as a 5′ end modificationor a 3′ end modification as described above. In certain embodiments, theend modification comprises dimethoxytrityl.

In certain embodiments, at least one of the modifications in thecombination comprises a modified base. In certain embodiments, themodified gRNA comprises 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, or 40 modified bases. In other embodiments,the modified gRNA comprises at least 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 110, 120, 130 or 140 modified bases. In certain embodiments, allbases in a gRNA are modified.

In certain embodiments, at least one of the modifications in thecombination comprises a modified sugar. In certain embodiments, themodified gRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, or 40 modified sugars. In other embodiments,the modified gRNA comprises at least 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 110, 120, 130 or 140 modified sugars. In certain embodiments, allsugars in a gRNA are modified.

In certain embodiments, at least one of the modifications in thecombination comprises a modified backbone (i.e., an internucleotidelinkage other than a natural phosphodiester). In certain embodiments,the modified-gRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, or 40 modified internucleotide linkages.In other embodiments, the modified gRNA comprises at least 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 110, 120, 130 or 140 modifiedinternucleotide linkages. In certain embodiments, all internucleotidelinkages in a gRNA are modified.

In certain embodiments, at least one of the modifications in thecombination comprises a 2′-O-methyl, a 2′-fluoro, a 2-amino, a 2′-deoxy,a 2′-arabino, a 2′-F-arabino, a 2-thiouracil, a 2-aminoadenine, a5-methylcytosine, a 5-aminoallyluracil, a Z base, a 3′-phosphorothioate,a 3′-phosphonoacetate, a 3′-phosphonoacetate ester, a3′-thiophosphonoacetate, a 3′-thiophosphonoacetate ester, a3′-methylphosphonate, a 3′-boranophosphonate, a 3′-phosphorodithioate,or combinations thereof. In certain embodiments, at least one of themodifications in the combination comprises a 2′-O-methyl, a 2′-deoxy, aZ base, a phosphorothioate internucleotide linkage, a phosphonoacetateinternucleotide linkage, a thiophosphonoacetate internucleotide linkage,or combinations thereof. In certain embodiments, at least one of themodifications in the combination comprises a 2′-F, a 2-thioU, a 4-thioU,a 2-aminoA, a 5-methylC, a 5-methylU, a 5-aminoallylU, or combinationsthereof. In certain embodiments, at least one of the modifications inthe combination is an “end” modification such as terminal phosphate, aPEG, a terminal amine, a terminal linker such as a hydrocarbon linker, asubstituted hydrocarbon linker, a squarate linker, a triazolo linker, aninternal linker such as 2-(4-butylamidofluorescein)propane-1,3-diolbis(phosphodiester) linker, a linker conjugated to a dye, a linkerconjugated to a non-fluorescent label, a linker conjugated to a tag or alinker conjugated to a solid support such as for example a bead ormicroarray. In certain embodiments, at least two of the modifications inthe combination comprise a 2′-O-methyl nucleotide and phosphorothioateinternucleotide linkage, a 2′-O-methyl nucleotide and phosphonoacetateinternucleotide linkage, or a 2′-O-methyl nucleotide andthiophosphonoacetate internucleotide linkage. In certain embodiments, atleast two of the modifications in the combination comprise a 2-O-methylnucleotide and phosphonocarboxylate internucleotide linkage, a2′-O-methyl nucleotide and phosphonocarboxylate ester internucleotidelinkage, a 2′-O-methyl nucleotide and thiophosphonocarboxylateinternucleotide linkage, a 2-O-methyl nucleotide andthiophosphonocarboxylate ester internucleotide linkage, or combinationsthereof. In other embodiments, the modifications in the combinationfurther comprise a 2-thiouracil, 2-thiocytosine, 4-thiouracil,6-thioguanine, 2-aminoadenine, 2-aminopurine, pseudouracil, inosine,7-deazaguanine, 7-deaza-8-azaguanine, 7-deazaadenine,7-deaza-8-azaadenine, 5-methylcytosine, 5-methyluracil,5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5,6-dehydrouracil,5-propynylcytosine, 5-propynyluracil, 5-ethynylcytosine,5-ethynyluracil, 5-allyluracil, 5-allylcytosine, 5-aminoallyl-uracil,5-aminoallyl-cytosine, or an abasic nucleotide.

In certain embodiments, at least one of the modifications in thecombination comprises a 2-O-methyl-3′-phosphorothioate. In certainembodiments, at least one of the modifications in the combinationcomprises a 2′-O-methyl-3-phosphonoacetate. In certain embodiments, atleast one of the modifications in the combination comprises a2′-O-methyl-3′-thiophosphonoacetate. In certain embodiments, at leastone of the modifications in the combination comprises a2′-halo-3′-phosphorothioate. In certain embodiments, at least one of themodifications in the combination comprises a2′-halo-3′-phosphonoacetate. In certain embodiments, at least one of themodifications in the combination comprises a2′-halo-3′-thiophosphonoacetate. In certain embodiments, at least one ofthe modifications in the combination comprises a2′-fluoro-3′-phosphorothioate. In certain embodiments, at least one ofthe modifications in the combination comprises a2′-fluoro-3′-phosphonoacetate. In certain embodiments, at least one ofthe modifications in the combination comprises a2′-fluoro-3′-thiophosphonoacetate. Possible combinations of at least twoor three modifications are represented in FIG. 6 and FIG. 7 respectivelyand are incorporated herein by reference.

In certain embodiments, the guide RNA comprises an oligonucleotiderepresented by Formula (III) or Formula (IV):

W-Y-Q   (III); or

Y-W-X-Q   (IV)

wherein Q and W each independently represent a nucleotide or a stretchof nucleotides of the oligonucleotide comprising at least onemodification and Y and X each independently represent an unmodifiedportion of the oligonucleotide.

In certain embodiments, W is within the 5′ portion of the guide RNA. Incertain embodiments, W is at least partially within the first five (5)nucleotides of the 5′ portion of the guide RNA. In certain embodiments,W is at least partially within the first three (3) nucleotides of the 5′portion of the guide RNA. In certain embodiments, W is within theinternal region (i.e., between the 5′ end and the 3′ end) of the guideRNA.

In certain embodiments, Q is within the 3′ portion of the guide RNA. Incertain embodiments, Q is at least partially within the last five (5)nucleotides of the 3′ portion of the guide RNA. In certain embodiments,Q is at least partially within the last three (3) nucleotides of the 3′portion of the guide RNA. In certain embodiments, Q is within theinternal region (i.e., between the 5′ end and the 3′ end) of the guideRNA.

In certain embodiments, W comprises an end modification as describedabove, such as a 5′ end or a 3′ end modification. In certainembodiments, the end modification comprises dimethoxytrityl.

In certain embodiments, at least one of W or Q comprises a modified baseas described above. In certain embodiments, at least one of W or Qcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 modifiedbases. In certain embodiments, at least one of W or Q comprises morethan one modified base, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19 or 20 modified bases.

In certain embodiments, at least one of W or Q comprises a modifiedsugar as described above. In certain embodiments, at least one of W or Qcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 modifiedsugars. In certain embodiments, at least one of W or Q comprises morethan one, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19 or 20 modified sugars.

In certain embodiments, at least one of W or Q comprises a modifiedbackbone (i.e., an internucleotide linkage other than a phosphodiester)as described above. In certain embodiments, at least one of W or Qcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 modifiedinternucleotide linkages. In certain embodiments, at least one of W or Qcomprises more than one, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19 or 20 modified internucleotide linkages.

In certain embodiments, at least one of W or Q comprises a 2′-O-methylnucleotide, a 2′-F nucleotide, a 2′-amino nucleotide, a 2′-deoxynucleotide, a 2-thiouridine nucleotide, a 2-aminoadenine nucleotide, a6-thioguanosine nucleotide, a 5-methylcytidine nucleotide, a5-aminoallyluridine nucleotide, a Z nucleotide, a 3′-phosphorothioateinternucleotide linkage, a 3′-phosphorothioate internucleotide linkage,a 3′-phosphonoacetate internucleotide linkage, a 3′-phosphonoacetateester internucleotide linkage, a 3′-thiophosphonoacetate internucleotidelinkage, a 3-thiophosphonoacetate ester internucleotide linkage, a3′-methylphosphonate internucleotide linkage, a 3′-boranophosphonateinternucleotide linkage, a 3′-phosphorodithioate internucleotidelinkage, or combinations thereof.

In certain embodiments, at least one of W or Q comprises a 2′-O-methyland a 3′-phosphorothioate group on the same nucleotide. In certainembodiments, at least one of W or Q comprises a 2′-O-methyl and a3′-phosphonoacetate group linkage on the same nucleotide. In certainembodiments, at least one of W or Q comprises a 2′-O-methyl and3′-thiophosphonoacetate group on the same nucleotide. In certainembodiments, at least one of W or Q comprises a 2′-F and a3′-phosphorothioate group on the same nucleotide. In certainembodiments, at least one of W or Q comprises a 2′-F and a3′-phosphonoacetate group linkage on the same nucleotide. In certainembodiments, at least one of W or Q comprises a 2′-F and3′-thiophosphonoacetate group on the same nucleotide.

In certain embodiments, at least one of W or Q comprises 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 modifiednucleotides. In certain embodiments, each of the modified nucleotideswithin at least one of W or Q comprises the same modification ormodifications. In certain embodiments, W comprises a modified nucleotidethat is different than a modified nucleotide in Q. In certainembodiments, at least one of W or Q comprises two or more modifiednucleotides. In certain embodiments, at least one of W or Q comprisesthree or more modified nucleotides. In certain embodiments, the modifiednucleotides are not arranged contiguously in the sequence, or at leastnot entirely, as one or more unmodified nucleotides may intercede. Incertain embodiments, the modified nucleotides are arranged contiguously.In certain embodiments, at least one of W or Q comprises at leastone-contiguous stretch of modified nucleotides. In certain embodiments,at least one of W or Q comprises a contiguous stretch of at least three(3) modified nucleotides. In certain embodiments, at least one of W or Qcomprises: a contiguous stretch of at least four (4) modifiednucleotides. In certain embodiments, at least one of W or Q comprises acontiguous stretch of at least five (5) modified nucleotides.

In certain embodiments, the guide RNA comprises a nucleotide sequence ofFormula (V) or Formula (VI):

M_(m)N_(n)M′_(m′)N′_(n′)  (Formula V); or

M_(m)N_(n)M′_(m′)N′_(n′)M″_(m″)  (Formula VI)

wherein each M independently represents a modified ribonucleotide;

wherein each N independently represents an unmodified ribonucleotide;

wherein each M′ independently represents a modified ribonucleotide;

wherein each N′ independently represents an unmodified ribonucleotide;

wherein each M″ independently represents a modified ribonucleotide;

wherein m is an integer between 0 and 40, n is an integer between 0 and130, m′ is an integer between 0 and 10, n′ is an integer between 0 and130, m″ is an integer between 0 and 10, provided that m+m′+m″ is greaterthan or equal to 1 and 50<m+n+m″+n″+m″≦150.

In certain embodiments, each M is independently selected from the groupconsisting of a 2-O-methyl ribonucleotide, a 2′-O-methyl-3′-P(S)ribonucleotide, a 2′-O-methyl-3-PACE ribonucleotide, a2′-O-methyl-3-thioPACE ribonucleotide, and a 2′-deoxynucleotide. Incertain embodiments, each M is independently selected from the groupconsisting of a 2′-O-methyl ribonucleotide, a 2′-O-methyl-3′-P(S)ribonucleotide, a 2′-O-methyl-3′-PACE ribonucleotide, and a2′-O-methyl-3′-thioPACE ribonucleotide. In certain embodiments, each Mis independently selected from the group consisting of a2′-O-methyl-3′-PACE ribonucleotide and a 2-O-methyl-3′-thioP ACEribonucleotide. In certain embodiments, where m >1, any given M is thesame or different from any other M. In certain embodiments, where m>1,each M comprises the same modification or modifications.

In certain embodiments, each M′ is independently selected from the groupconsisting of a 2′-O-methyl ribonucleotide, a 2′-O-methyl-3′-P(S)ribonucleotide, a 2′-O-methyl-3′-PACE ribonucleotide, a2′-O-methyl-3′-thioPACE ribonucleotide, and a 2′-deoxynucleotide. Incertain embodiments, each M′ is independently selected from the groupconsisting of a 2′-O-methyl ribonucleotide, a 2′-O-methyl-3′-P(S)ribonucleotide, a 2′-O-methyl-3′-PACE ribonucleotide, and a2′-O-methyl-3′-thioPACE ribonucleotide. In certain embodiments, each M′is independently selected from the group consisting of a2′-O-methyl-3′-PACE ribonucleotide and a 2′-O-methyl-3′-thioPACEribonucleotide. In certain embodiments, where m′>1, any given M′ is thesame or different from any other M In certain embodiments, where m′>1,each M′ comprises the same modification or modifications.

In certain embodiments, each M″ is independently selected from the groupconsisting of a 2′-O-methyl ribonucleotide, a 2′-O-methyl-3l-P(S)ribonucleotide, a 2′-O-methyl-3′-PACE ribonucleotide, a2′-O-methyl-3-thioPACE ribonucleotide, and a 2′-deoxynucleotide. Incertain embodiments, each M″ is independently selected from the groupconsisting of a 2-O-methyl ribonucleotide, a 2′-O-methyl-3′-P(S)ribonucleotide, a 2′-O-methyl-3′-PACE ribonucleotide, and a2′-O-methyl-3-thioPACE ribonucleotide. In certain embodiments, each M″is independently selected from the group consisting of a2′-O-methyl-3′-PACE ribonucleotide and a 2-O-methyl-3-thioPACEribonucleotide. In certain embodiments, where m″>1, any given M″ is thesame or different from any other M″. In certain embodiments, where m″>1,each M″ comprises the same modification or modifications.

In certain embodiments, each M is a 2′-O-methyl-3′-PACE ribonucleotide;m is selected from an integer between 1 and 10; each N is independentlyselected from the group consisting of A, U, C, and G; n is selected froman integer between 10 and 130; each M′ is independently selected fromthe group consisting of a 2′-O-methyl ribonucleotide, a2′-O-methyl-3′-P(S)ribonucleotide, a 2′-O-methyl-3′-PACE ribonucleotide,a 2′-O-methyl-3′-thioPACE ribonucleotide, a 2′-deoxynucleotide, and a Znucleotide; m′ is selected from an integer between 1 and 10; each N isindependently selected from the group consisting of A, U, C, and G; andn′ is selected from an integer between 0 and 130. In certainembodiments, each M′ is a 2′-O-methyl-3′-PACE ribonucleotide. In certainembodiments, each M′ is a 2′-O-methyl-3′-thioPACE ribonucleotide. Incertain embodiments, each M′ is a 2′-O-methyl ribonucleotide. In certainembodiments, each M′ is a 2′-O-methyl-3′-P(S) ribonucleotide. In certainembodiments, each M′ is a Z nucleotide.

In certain embodiments, each M is a 2′-O-methyl-3′-thioPACEribonucleotide; m is selected from an integer between 1 and 10; each Nis independently selected from the group consisting of A, U, C, and G; nis selected from an integer between 10 and 130; each M′ is independentlyselected from the group consisting of a 2′-O-methyl ribonucleotide, a2′-O-methyl-3′-P(S) ribonucleotide, a 2′-O-methyl-3′-PACEribonucleotide, a 2′-O-methyl-3′-thioPACE ribonucleotide, a2′-deoxynucleotide, and a Z nucleotide; m′ is selected from an integerbetween 1 and 10; each N is independently selected from the groupconsisting of A, U, C, and G; and nr is selected from an integer between0 and 130. In certain embodiments, each M is a 2′-O-methyl-3′-PACEribonucleotide. In certain embodiments, each M′ is a2′-O-methyl-3′-thioPACE ribonucleotide. In certain embodiments, each M′is a 2′-O-methyl ribonucleotide. In certain embodiments, each M′ is a2′-O-methyl-3′-P(S) ribonucleotide. In certain embodiments, each M′ is aZ nucleotide.

In certain embodiments, each M is a 2′-O-methyl-3′-P(S) ribonucleotide;m is selected from an integer between 1 and 10; each N is independentlyselected from the group consisting of A, U, C, and G; n is selected froman integer between 10 and 130; each M′ is independently selected fromthe group consisting of a 2-O-methyl ribonucleotide, a2′-O-methyl-3′-P(S) ribonucleotide, a 2′-O-methyl-3-PACE ribonucleotide,a 2′-O-methyl-3′-thioPACE ribonucleotide, a 2′-deoxynucleotide, and a Znucleotide; m′ is selected from an integer between 1 and 10; each N isindependently selected from the group consisting of A, U, C, and G; andn′ is selected from an integer between 0 and 130. In certainembodiments, each M′ is a 2′-O-methyl-3′-PACE ribonucleotide. In certainembodiments, each M′ is a 2′-O-methyl-3′-thioPACE ribonucleotide. Incertain embodiments, each M′ is a 2′-O-methyl ribonucleotide. In certainembodiments, each M′ is a 2′-O-methyl-3′-P(S) ribonucleotide. In certainembodiments, each M′ is a Z nucleotide.

In certain embodiments, each M is independently selected from the groupconsisting of a 2-O-methylribonucleotide, a 2′-O-methyl-3′-P(S)ribonucleotide, a 2′-O-methyl-3′-PACE ribonucleotide, a2′-O-methyl-3′-thioPACE ribonucleotide, a 2′-deoxynucleotide, and a Znucleotide; hi is selected from an integer between 0 and 10; each N isindependently selected from the group consisting of A, U, C, and G; n isselected from an integer between 10 and 15; each M′ is a 2′-O-methylribonucleotide; m′ is selected from an integer between 1 and 5; each Nis independently selected from the group consisting of A, U, C, and G;and n′ is selected from an integer between 0 and 130. In certainembodiments, each M is a 2′-O-methyl-3′-PACE ribonucleotide. In certainembodiments, each M is a 2′-O-methyl-3′-thioPACE ribonucleotide. Incertain embodiments, each M is a 2′-O-methyl ribonucleotide. In certainembodiments, each M is a 2′-O-methyl-3′-P(S)ribonucleotide. In certainembodiments, m is 0; n is selected from an integer between 10 and 15, m′is selected from an integer between 1 and 5; and n′ is selected from aninteger between 0 and 130.

In certain embodiments, at least one of the modifications in thecombination is a stability-altering modification. In certainembodiments, at least one of the modifications in the combinationincreases nuclease resistance of the guide RNA relative to a guide RNAwithout the modification, thus it enhances the stability of the guideRNA.

In certain embodiments, at least one of the modifications in thecombination is a stability-enhancing modification as described above.

In certain embodiments, at least one of the modifications in thecombination is a specificity-altering modification as described above.

In certain embodiments, at least one of the modifications in thecombination alters RNA base pairing. Tn certain embodiments, at leastone of the modifications in the combination lowers the Tm of a basepairing interaction as described above. In certain embodiments, at leastone of the modifications in the combination raises the Tm of a basepairing interaction as described above.

In certain embodiments, at least one of the modifications in thecombination alters transfection efficiency of the guide RNA relative toa guide RNA without the modification. In certain embodiments, at leastone of the modifications in the combination increases transfectionefficiency of the guide RNA relative to a guide RNA without themodification. In certain embodiments, at least one of the modificationsin the combination decreases transfection efficiency of the guide RNArelative to a guide RNA without the modification. In certainembodiments, at least one of the transfection-increasing modificationsin the combination comprises a phosphonoacetate alkyl esterinternucleotide linkage, such as a phosphonoacetate methyl esterinternucleotide linkage.

In certain embodiments, at least one of the modifications in thecombination enhances stability and specificity of the guide RNA relativeto a guide RNA without the modification. In certain embodiments, atleast one of the modifications in the combination enhances stability andtransfection efficiency of the guide RNA relative to a guide RNA withoutthe modification. In certain embodiments, at least one of themodifications in the combination enhances specificity and transfectionefficiency of the guide RNA relative to a,guide RNA without themodification.

In certain embodiments, at least one of the modifications in thecombination alters the secondary structure of the guide RNA. Thismodification alters the base-pairing of any of the RNA/RNA internalduplexes in the guide RNA. Some of these modifications increase the basepairing of the RNA/RNA structure or alternatively increase the Tm of theRNA/RNA duplex, whereas other modifications decrease the base pairing(or Tm) of the RNA/RNA duplex or duplexes. Such modifications includebase modified nucleotides, particularly UNA nucleotides such as the2-thiouridine and 2-aminoadenosine pair, the Z/P nucleotide pair, theisoC/isoG pair, the 6-thioG/5-methylpyrimidine pair, and nucleotideswith modifications on the sugar or the internucleotide linkages asdiscussed before.

In certain embodiments, the combination includes at least onemodification or a set of modifications that increases nucleasesresistance (i.e., stability) with at least one modification or a set ofmodifications that increases specificity (i.e., reduces off-targeteffects). In certain embodiments, the combination includes at least onemodification or a set of modifications that increases nucleasesresistance (i.e., stability) with at least one modification or a set ofmodifications that raises the Tm of some bases pairing in the guide RNA.In certain embodiments, the combination includes at least onemodification or a set of modifications that increases nucleasesresistance (i.e., stability) with at least one modification or a set ofmodifications that lowers the Tm of some bases pairing of the guide RNA.In certain embodiments, the combination includes at least onemodification or a set of modifications that increases nucleaseresistance (i.e., stability), at least one modification or a set ofmodifications that increases the Tm of some bases paring in the guideRNA, and at least one modification or a set of modifications thatdecreases the Tm of some base paring elsewhere in the guide RNA. Incertain embodiments the combination includes at least one modificationor a set of modifications that increases nuclease resistance (i.e.,stability) and at least one modification or a set of modifications thatincreases the binding of the guide RNA to Gas protein. In certainembodiments, the combination includes at least one modification or a setof modifications that increases nuclease resistance (i.e., stability)and at least one modification or a set of modifications that decreasesthe binding of the guide RNA to Cas protein. In certain embodiments, theguide RNA comprises a combination of the different types ofmodifications.

D. Guide RNA Structure

In certain embodiments, the guide RNA is able to form a complex with aCRISPR-associated-protein. In certain embodiments, the CRISPR-associatedprotein is provided by or is derived from a CRISPR-Cas type II system,which has an RNA-guided polynucleotide binding and/or nuclease activity.In certain embodiments, the CRISPR-associated protein is Cas9, a Cas9mutant, or a Cas9 variant. In certain embodiments, the CRISPR-associatedprotein is the Cas9 nuclease from Streptococcus pyogenes. In certainembodiments, the CRISPR-associated protein is the Cas9 nuclease fromStreptococcus thermophilus. In certain embodiments, theCRISPR-associated protein is the Cas9 nuclease from Staphylococcusaureus. In certain embodiments, the synthetic guide RNA or a syntheticguide RNA:CRISPR-associated protein complex maintains functionality ofnatural guide RNA or a complex that does not have modified nucleotides.In certain embodiments, the functionality includes binding a targetpolynucleotide. In certain embodiments, the functionality includesnicking a target polynucleotide. In certain embodiments, thefunctionality includes cleaving a target polynucleotide. In certainembodiments, the target polynucleotide is within a nucleic acid invitro. In certain embodiments, the target polynucleotide is within thegenome of a cell in vivo or in vitro (such as in cultured cells or cellsisolated from an organism). In certain embodiments, the targetpolynucleotide is a protospacer in DNA.

In certain embodiments, the crRNA segment comprises from 25 to 80nucleotides. In certain embodiments, the crRNA segment comprises a guidesequence that is capable of hybridizing to a target sequence. In certainembodiments, the guide sequence is complementary to the target sequenceor a portion thereof. In certain embodiments, the guide sequencecomprises from 15 to 30 nucleotides. In certain embodiments, the crRNAsegment comprises a stem sequence. In certain embodiments, the stemsequence comprises from 10 to 50 nucleotides. In certain embodiments,the crRNA segment comprises a 5′-overhang sequence. In certainembodiments, the 5′-overhang sequence comprises from 1 to 10nucleotides, alternatively 1 to 5 nucleotides, alternatively 1, 2 or 3nucleotides. In certain embodiments, the crRNA comprises both (i) aguide sequence that is capable of hybridizing to a target sequence and(ii) a stem sequence. In certain embodiments, the crRNA comprises (i) a5′-overhang sequence, (ii) a guide sequence that is capable ofhybridizing to a target sequence, and (iii) a stem sequence. In certainembodiments wherein the crRNA segment comprises a stem sequence, thetracrRNA segment comprises a nucleotide sequence that is partially orcompletely complementary to the stem sequence of the crRNA segment. Incertain embodiments, the tracrRNA segment comprises at least one moreduplex structure. In certain embodiments, the guide RNA is a singleguide RNA. In certain embodiments, the guide RNA is a single guide RNA,wherein the crRNA segment and the tracrRNA segment are linked through aloop L. In certain embodiments, the loop L comprises 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 nucleotides. In certain embodiments, the loop L comprisesa nucleotide sequence of GNRA, wherein N represents A, C, G, or U and Rrepresents A or G. In certain embodiments, the loop L comprises anucleotide sequence of GAAA. In certain embodiments, the guide RNAcomprises more than one loop.

The guide RNA comprises a 5′ portion (i.e., the 5′ half) and a 3′portion (i.e., the 3′ half). In certain embodiments, the crRNA segmentis 5′ (i.e., upstream) of the tracrRNA segment. In certain embodiments,the tracrRNA segment is 5′ relative to the crRNA segment.

In certain embodiments, the guide RNA comprises at least two separateRNA strands, for example, a crRNA strand and a separate tracrRNA strand.See, for example, FIG. 5A. In certain embodiments, each of the strandsis a synthetic strand comprising one or more modifications. In certainembodiments, at least one of the strands is a synthetic strandcomprising one or more modifications. In certain embodiments, thestrands function together to guide binding, nicking, or cleaving of atarget polynucleotide by a Cas protein, such as Cas9. In certainembodiments, the crRNA sequence and the tracrRNA sequence are onseparate stands and hybridize to each other via two complementarysequences to form a stem or duplex.

In certain embodiments, the guide RNA is a single guide, RNA comprisinga crRNA sequence and a tracrRNA sequence. See, for example, FIG. 5B. Incertain embodiments, the crRNA sequence and the tracrRNA sequence areconnected by a loop sequence or “loop.” In certain embodiments, a singleguide RNA comprises a 5′ portion and a 3′ portion, wherein the crRNAsequence is upstream of the tracrRNA sequence.

In certain embodiments, the total length of the two RNA pieces can beabout 50-220 (e.g., about 55-200, 60-190, 60-180, 60-170, 60-160,60-150, 60-140, 60-130, and 60-120) nucleotides in length, such as about60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,or 220 nucleotides in length. Similarly, the single guide RNA (e.g.,FIG. 5B) can be about 50-220 (e.g., about 55-200, 60-190, 60-180,60-170, 60-160, 60-150, 60-140, 60-130, and 60-120) nucleotides inlength, such as about 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, or 220 nucleotides in length.

As shown in FIGS. 5A and 5B, the synthetic guide RNA comprises (i) acrRNA sequence that comprises (a) a guide sequence (e.g., segmentG₁-G_(n), where each G represents a nucleotide in the guide sequence)capable of hybridizing to a target sequence in a nucleic acid, (b) afirst stem sequence (e.g., segment X₁-X_(n), where each X represents anucleotide in the first-stem sequence) capable of hybridizing partiallyor completely to a second stem sequence, and, optionally (c) a 5′overhang sequence (e.g., segment O₁-O_(n), where each O represents anucleotide in the overhang sequence), and (ii) a tracrRNA sequence thatcomprises the second stem sequence (e.g., segment Y₁-Y_(n), where each Yrepresents a nucleotide in the second stem sequence). The tracrRNAsequence further comprises segment T₁-T_(n), where each T represents anucleotide in the tracrRNA sequence. The synthetic guide RNA shown inFIG. 5A includes one or more modifications. Likewise, the syntheticguide RNA shown in FIG. 5B includes one or more modifications. Incertain embodiments, the modification is located at any point along thelength of the crRNA, the tracrRNA, or the single guide RNA comprising acrRNA segment, a tracrRNA segment, and, optionally, a loop. In certainembodiments, any nucleotide represented by O, G, X, Y, or T in thesynthetic guide RNA shown in FIGS. 5A and 5B may be a modifiednucleotide. The guide RNA shown in FIG. 5B represents a single guide RNA(sgRNA) where the crRNA segment and the tracrRNA segment are connectedby a loop having the sequence GNRA, wherein N represents A, C, G, or U,and R represents A or G.

In certain embodiments, the crRNA segment of the guide RNA is 25-70(e.g., 30-60, 35-50, or 40-45) nucleotides in length. In certainembodiments, the guide sequence is 12-30 (e.g., 16-25, 17-20, or 15-18)nucleotides in length. In some embodiments, a 5′ portion of the crRNAdoes not hybridize or only partially hybridizes with the targetsequence. For example, there can be a 5′-overhang on the crRNA segment.

In certain embodiments, the single guide RNA comprises a central portionincluding the stem sequence of the crRNA segment, the stem sequence ofthe tracrRNA segment, and, optionally, a loop that covalently connectsthe crRNA segment to the tracrRNA segment. In certain embodiments, thecentral segment of the single guide RNA is 8-60 (e.g., 10-55, 10-50, or20-40) nucleotides in length.

In certain embodiments, the tracrRNA segment of the guide RNA is 10-130(e.g., 10-125, 10-100, 10-75, 10-50, or 10-25) nucleotides in length. Incertain embodiments, the tracrRNA segment includes one or more hairpinor duplex structures in addition to any hairpin or duplex structure inthe central segment.

In certain embodiments, the tracrRNA is truncated compared to areference tracrRNA, such as a naturally existing mature tracrRNA. Arange of lengths has been shown to function in both the separate type(FIG. 5A) and the chimeric sgRNA type (FIG. 5B). For example, in certainembodiments, tracrRNA may be truncated from its 3′ end by at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 or 40 nts. In certainembodiments, the tracrRNA molecule may be truncated from its 5′ end byat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75 or 80 nts. In certain embodiments, the tracrRNAmolecule may be truncated from both the 5′ and 3′ end, e.g., by at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 nts from the 5′ end and at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 or 40 nts from the 3′end. See, e.g., Jinek et al. (2012) Science, 337, 816-21; Mali et al.(2013) Science, 339:6121, 823-6; Cong et al. (2013) Science, 339:6121,819-23; and Hwang et al. (2013) Nat. Biotechnol. 31:3, 227-9; Jinek etal. (2013) eLife, 2, e00471. In certain embodiments, the tracrRNA isuntruncated.

In certain embodiments, the disclosed modifications we in the crRNAsegment or the tracrRNA segment or both. In certain embodiments, thedisclosed modifications are in the guide sequence of the crRNA segment.In certain embodiments, the disclosed modifications are in the stemsequence of the crRNA segment. In certain embodiments, the disclosedmodifications are in the 5-overhang sequence of the crRNA segment. Incertain embodiments, the disclosed modifications are in the stemsequence of the tracrRNA segment. In certain embodiments, the disclosedmodifications are in the loop sequence of the guide RNA. In certainembodiments, the disclosed modifications are in the 51 portion of theguide RNA. In certain embodiments, the disclosed modifications are inthe 3′ portion of the guide RNA. In certain embodiments, the disclosedmodifications are in the 5′ portion of the guide RNA and the 3′ portionof the guide RNA.

E. Synthesis of Guide RNA

In certain embodiments, guide RNAs, including single guide RNAs (sgRNAs;see FIGS. 1 and 5B) are produced by chemical synthesis using the art ofsynthetic organic chemistry. A guide RNA that comprises any nucleotideother than the four predominant ribonucleotides, namely A, C, G, and U,whether unnatural or natural, such as a pseudouridine, inosine or adeoxynucleotide, possesses a chemical modification or substitution atthe nucleotide which is chemically/structurally distinct from any of thefour predominant nucleotides in RNAs.

The synthetic guide RNAs described herein can be chemically synthesized.For example, the synthetic guide RNAs can be synthesized using TCchemistry by th& method described in Dellinger et al, (2011) J. Am.Chem. Soc., 133, 11540, U.S. Pat. No. 8,202,983, and US PatentApplication 2010/0076183A1, the contents of which are incorporated byreference in their entireties. “TC chemistry” refers to the compositionand methods of using RNA monomeric nucleotide precursors protected onthe 2′-hydroxyl moiety by a thionocarbamate protecting group, tosynthesize unmodified RNA or modified RNA comprising one or moremodified nucleotides. The ability to chemically synthesize relativelylong RNAs (as long as 200 mers or more) using TC-RNA chemistry allowsone to produce guide RNAs with special features capable of outperformingthose enabled by the four predominant ribonucleotides (A, C, G and U).Some synthetic guide RNAs described herein can also be made usingmethods known in the art that include in vitro transcription andcell-based expression. For example, 2′-fluoro NTPs can be incorporatedinto synthetic guide RNAs produced by cell-based expression.

Synthesis of guide RNAs can also be accomplished by chemical orenzymatic synthesis of RNA sequences that are subsequently ligatedtogether by enzymes, or chemically ligated by chemical ligation,including but not limited to cyanogen bromide chemistry, “click”chemistry as published by R. Kumar et al. (2007) J. Am. Chem. Soc., 129,6859-64, or squarate conjugation chemistry as described by K. Hill inWO2013176844 titled “Compositions and methods for conjugatingoligonucleotides.”

As further described below, a guide RNA disclosed herein, includingthose comprising modified nucleotides and/or modified internucleotidelinkages, can be used to perform various CRISPR-mediated functions(including but not limited to editing genes, regulating gene expression,cleaving target sequences, and binding to target sequences) in vitro orin v/vo, such as in cell-free assays, in intact cells, or in wholeorganisms. For in vitro or in vivo applications, the RNA can bedelivered into cells or whole organisms in any mannerkriown in the art.

Libraries and Arrays

In one aspect, the present invention provides a set or library ofmultiple guide RNAs. In certain embodiments, the library contains two ormore guide RNAs disclosed herein: The library can contain from about 10to about 10⁷ individual members, e.g., about 10 to about 10², about 10²to about 10³, about 10³ to about 10⁵, from about 10⁵ to about 10⁷members. An individual member of the library differs from other membersof the library at least in the guide sequence, i.e., the DNA targetingsegment of the gRNA. On the other hand, in certain embodiments, eachindividual member of a library can contain the same or substantially thesame nucleotide sequence for the tracrRNA segment as all the othermembers of the library. In this way, the library can comprise membersthat target different polynucleotides or different sequences in one ormore polynucleotides.

In certain embodiments, the library comprises at least 10² unique guidesequences. In certain embodiments, the library comprises at least 10³unique guide sequences. In certain embodiments, the library comprises atleast 10⁴ unique guide sequences. In certain embodiments, the librarycomprises at least 10⁵ unique guide sequences. In certain embodiments,the library comprises at least 10⁶ unique guide sequences. In certainembodiments, the library comprises at least 10⁷ unique guide sequences.In certain embodiments, the library targets at least 10 differentpolynucleotides. In certain embodiments, the library targets at least10² different polynucleotides. In certain embodiments, the librarytargets at least 10³ different polynucleotides. In certain embodiments,the library targets at least 10⁴ different polynucleotides. In certainembodiments, the library targets at least 10⁵ different polynucleotides.In certain embodiments, the library targets at least 10⁶ differentpolynucleotides. In certain embodiments, the library targets at least10⁷ different polynucleotides.

In certain embodiments, the library comprises a collection of guide RNAshaving the same sequence and the same, modifications in a progressivelyshifted window that moves across the sequence of the members in thelibrary. In certain embodiments, the windows collectively cover theentire length of the RNA.

In certain embodiments, the library allows one to conducthigh-throughput, multi-target genomic manipulations and analyses. Incertain embodiments, only the DNA-targeting segments of the guide RNAsare varied, while the Cas protein-binding segment is the same. Incertain embodiments, a first portion of the library comprises guide RNAspossessing a Cas-binding segment that recognizes, binds and directs aparticular Cas protein and a second portion of the library comprises adifferent Cas-binding segment that recognizes, binds and directs adifferent Cas protein (e.g., a Cas protein from a different species),thereby allowing the library to function with two or more orthogonal Casproteins. In certain embodiments, induced expression of a firstorthogonal Cas protein utilizes the portion of the library whichinteracts with the first orthogonal Cas protein. In certain embodiments,induced expression of a first and second orthogonal Cas protein utilizesthe portions of the library which interact with the first and secondorthogonal Cas proteins, respectively. In certain embodiments, inducedexpression of the first, and second orthogonal Cas proteins occur atdifferent times. Accordingly, one can carry out large-scale gene editingor gene regulation by specifically manipulating or modifying multipletargets as specified in the library.

In certain embodiments, the library is an “arrayed” library, namely acollection of different features or pools of features in an addressablearrangement. For example, features of an array can be selectivelycleaved and transferred to a microtiter plate such that each well in theplate contains a known feature or a known pool of features. In someother embodiments, the library is synthesized in a 48-columns or in a96-columns microtiter plate format or in a 384-columns plate.

In certain embodiments, synthesis of the guide RNA of this invention maybe conducted on a solid support having a surface to which chemicalentities may bind. In some embodiments, guide RNAs being synthesized areattached, directly or indirectly, to the same solid support and may formpart of an array. An “array” is a collection of separate molecules ofknown monomelic sequence each arranged in a spatially defined and aphysically addressable manner, such that the location of each sequenceis known. An “array,” or “microarray” used interchangeably hereinincludes any one-dimensional, two-dimensional or substantiallytwo-dimensional (as well as a three-dimensional) arrangement ofaddressable regions bearing a particular chemical moiety or moieties(such as ligands, e.g., biopolymers such as polynucleotide oroligonucleotide sequences (nucleic acids), polypeptides (e.g.,proteins), carbohydrates, lipids, etc.) associated with that region. Anarray is “addressable” when it has multiple regions of differentmoieties (e.g., different polynucleotide sequences) such that a region(i.e., a “feature” of the array) at a particular predetermined location(i.e., an “address”) on the array will detect a particular target orclass of targets (although a feature may incidentally detect non-targetsof that feature). Array features are typically, but need not be,separated by intervening spaces. The number of features that can becontained on an array will largely be determined by the surface area ofthe substrate, the size of a feature and the spacing between features.Arrays can have densities of up to several hundred thousand or morefeatures per cm², such as 2,500 to 200,000 features/cm². The featuresmay or may not be covalently bonded to the: substrate.

Suitable solid supports may have a variety of forms and compositions andderive from naturally occurring materials, naturally occurring materialsthat have been synthetically modified, or synthetic materials. Examplesof suitable support materials include, but are not limited to, silicas,silicon and silicon oxides, teflons, glasses, polysacchandes such asagarose (e.g., Sepharose(r) from Pharmacia) and dextran (e.g.,Sephadex(r) and Sephacyl(r), also from Pharmacia), polyacrylamides,polystyrenes, polyvinyl alcohols, copolymers of hydroxyethylmethacrylate and methyl methacrylate, and the like. In some embodiments,the solid support is a plurality of beads.

The initial monomer of the guide RNAs to be synthesized on the substratesurface can be bound to a linker which in turn is bound to a surfacehydrophilic group, e.g., a surface hydroxyl moiety present on a silicasubstrate. In some embodiments, a universal linker is used. In someother embodiments, the initial monomer is reacted directly with, e.g., asurface hydroxyl moiety. Alternatively, guide RNAs can be synthesizedfirst according to the present invention, and attached to a solidsubstrate post-synthesis by any method known in the art. Thus, thepresent invention can be used to prepare arrays of guide RNAs, whereinthe oligonucleotides are either synthesized on the array, or attached tothe array substrate post-synthesis. Subsequently, the guide RNAs or apool or a plurality of pools of guide RNAs can optionally andselectively be cleaved from the array substrate and be used as a libraryor libraries.

IV. Cas Proteins

As mentioned above, a functional CRISPR-Cas system also requires aprotein component (e.g., a Cas protein, which may be a Cas nuclease)that provides a desired activity, such as target binding or targetnicking/cleaving. In certain embodiments, the desired activity is targetbinding. In certain embodiments, the desired activity is target nickingor target cleaving. In certain embodiments, the desired activity alsoincludes a function provided by a polypeptide that is covalently fusedto a Cas protein, as disclosed herein. In certain embodiments, thedesired activity also includes a function provided by a polypeptide thatis covalently fused to a nuclease-deficient Cas protein, as disclosedherein. Examples of such a desired activity include a transcriptionregulation activity (either activation or repression), an epigeneticmodification activity, or a target visualization/identificationactivity, as described below. The Cas protein can be introduced into anin vitro or in vivo system as a purified or non-purified (i) Cas proteinor (ii) mRNA encoded for expression of the Cas protein or (iii) linearor circular DNA encoded for expression of the protein. Any of these 3methods of providing the Cas protein are well known in the art and areimplied interchangeably when mention is made herein of a Cas protein oruse of a Gas protein. In certain embodiments, the Cas protein isconstitutively expressed from mRNA or DNA. In certain embodiments, theexpression of Cas protein from mRNA Or DNA is inducible or induced.

In certain embodiments, the Cas protein is chemically synthesized (seee.g., Creighton, “Proteins: Structures and Molecular Principles,” W. H.Freeman & Co., NY, 1983), or produced by recombinant DNA technology asdescribed herein. For additional guidance, skilled artisans may consultFrederick M. Ausubel et al., “Current Protocols in Molecular Biology,”John Wiley & Sons, 2003; and Sambrook et al., “Molecular Cloning, ALaboratory Manual,” Cold Spring Harbor Press, Cold Spring Harbor, N.Y.,2001).

In certain embodiments, the Cas protein is provided in purified orisolated form. In certain embodiments, the Cas protein is provided atabout 80%, about 90%, about 95%, or about 99% purity. In certainembodiments, the Cas protein is provided as part of a composition. Incertain embodiments, the Cas protein is provided in aqueous compositionssuitable for use as, or inclusion in, a composition for an RNA-guidednuclease reaction. Those of skill in the art are well aware of thevarious substances that can be included in such nuclease reactioncompositions.

In certain embodiments, a Cas protein is provided as a recombinantpolypeptide. In certain examples, the recombinant polypeptide isprepared as a fusion protein. For example, in certain embodiments, anucleic acid encoding the Cas protein is linked to another nucleic acidencoding a fusion partner, glutathione-s-transferase (GST), 6x-Hisepitope tag, or M13 Gene 3 protein. Suitable host cells can be used toexpresses the fusion protein. In certain embodiments, the fusion proteinis isolated by methods known in the art. In certain embodiments, thefusion protein can be further treated, e.g., by enzymatic digestion, toremove the fusion partner and obtain the Cas protein. Alternatively, Casprotein:guide RNA complexes can be made with recombinant technologyusing a host cell system or an in vitro translation-transcription systemknown in the art. Details of such systems and technology can be found ine.g., WO2014144761 WO2014144592, WO2013176772, US20140273226, andUS20140273233, the contents of which are incorporated herein byreference in their entireties.

Wild Type Cas Proteins

In certain embodiments, a Cas protein comprises a protein derived from aCRISPR-Cas type I, type II, or type III system, which has an RNA-guidedpolynucleotide binding and/or nuclease activity. Non-limiting examplesof suitable Cas proteins include Cas3, Cas4, Cas5, Cas5e (or CasD),Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10,Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (orCasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2,Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3,Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3,Csf4, and Cu1966. See e.g., WO2014144761 WO2014144592, WO2013176772,US20140273226, and US20140273233, the contents of which are incorporatedherein by reference in their entireties.

In certain embodiments, the Cas protein is derived from a type IICRISPR-Cas system. In certain embodiments, the Cas protein is or isderived from a Cas9 protein. In certain embodiments, the Cas protein isor is derived from a bacterial Cas9 protein, including those identifiedin WO2014144761. In certain embodiments, the Cas protein is or isderived from a Streptococcus sp. or Staphylococcus sp. Cas9 protein. Incertain embodiments, the Cas protein is or is derived from theStreptococcus thermophilus Cas9 protein. In certain embodiments, the Casprotein is or is derived from a the Streptococcus pyogenes Cas9 protein.In certain embodiments, the Cas protein is or is derived from theStaphylococcus aureus Cas9 protein. In certain embodiments, the Casprotein is or is derived from the Streptococcus thermophilus Cas9protein.

In certain embodiments, the wild type Cas protein is a Cas9 protein. Incertain embodiments, the wild type Cas9 protein is the Cas9 protein fromS. pyogenes (SEQ ID NO: 1). In certain embodiments, the protein orpolypeptide can comprise, consist of, or consist essentially of afragment of SEQ ID NO: 1.

In general, a Cas protein includes at least one RNA binding domain,which interacts with the guide RNA. In certain embodiments, the Casprotein is modified to increase nucleic acid binding affinity and/orspecificity, alter an enzymatic activity, and/or change another propertyof the protein. For example, nuclease (i.e., DNase, RNase) domains ofthe Cas protein can be modified, mutated, deleted, or inactivated.Alternatively, the Cas protein can be truncated to remove domains thatare not essential for the function of the protein. In certainembodiments, the Cas protein is truncated or modified to optimize theactivity of the effector domain. In certain embodiments, the Cas proteinincludes a nuclear localization sequence (NLS) that effects importationof the NLS-tagged Cas protein into the nucleus of a living cell. Incertain embodiments, the Cas protein includes two or more modifications.

Mutant Cas Proteins

In some embodiments, the Cas protein-can be a mutant of a wild type Casprotein (such as Cas9) or a fragment thereof. In other embodiments, theCas protein can be derived from a mutant Cas protein. For example, theamino acid sequence of the Cas9 protein can be modified to alter one ormore properties (e.g., nuclease activity, binding affinity, stability toproteases, etc.) of the protein. Alternatively, domains of the Cas9protein not involved in RNA-guided cleavage can be eliminated from theprotein such that the modified Cas9 protein is smaller than the wildtype Cas9 protein. For example, reducing the size of the Cas9 codingsequence can allow it to fit within a transfection vector that otherwisecannot accommodate the wild type sequence, such as the AAV vector amongothers. In some embodiments, the present system utilizes the Cas proteinfrom S. pyogenes, either as encoded in bacteria or codon-optimized forexpression in eukaryotic cells. Shown below is the amino acid sequenceof wild type S. pyogenes Cas9 protein sequence (SEQ ID No. 1, availableat www.uniprot.org/uniprot/Q99ZW2).

MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEWKFCMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

A Cas9 protein generally has at least two nuclease e.g., DNase) domains.For example, a Cas9 protein can have a RuvC-like nuclease domain and anHNH-like nuclease domain. The RuvC and HNH domains work together to cutboth strands in a target site to make a double-stranded break in thetarget polynucleotide. (Jinek et al., Science, 337: 816-821). In certainembodiments, a mutant Cas9 protein is modified to contain only onefunctional nuclease domain (either a RuvC-like or an HNH-like nucleasedomain). For example, in certain embodiments, the mutant Cas9 protein ismodified such that one of the nuclease domains is deleted or mutatedsuch that it is no longer functional (i.e., the nuclease activity isabsent). In some embodiments where one of the nuclease domains isinactive, the mutant is able to introduce a nick into a double-strandedpolynucleotide (such protein is termed a “nickase”) but not able tocleave the double-stranded polynucleotide. For example, an aspartate toalanine (D10A) conversion in a RuvC-like domain converts theCas9-derived protein into a nickase. Likewise, a histidine to alanine(H840A) conversion in a HNH domain converts the Cas9-derived proteininto a nickase. Likewise, an asparagine to alanine (N863A) conversion ina HNH domain converts the Cas9-derived protein into a nickase.

In certain embodiments, both the RuvC-like nuclease domain and theHNH-like nuclease domain arc modified or eliminated such that the mutantCas9 protein is unable to nick or cleave the target polynucleotide. Incertain embodiments, all nuclease domains of the Cas9-derived proteinare modified or eliminated such that the Cas9-derived protein lacks allnuclease activity. In certain embodiments, a Cas9 protein that lackssome or all nuclease activity relative to a wild-type counterpart,nevertheless, maintains target recognition activity to a greater orlesser extent.

In any of the above-described embodiments, any or all of the nucleasedomains can be inactivated by one or more deletion mutations, insertionmutations, and/or substitution mutations using well-known methods, suchas site-directed mutagenesis, PCR-mediated mutagenesis, and total genesynthesis, as well as other methods known in the art.

In certain embodiments, the “Cas mutant” or “Cas variant” is at least50% (e.g., any number between 50% and 100%, inclusive, e.g., 50%, 60%,70%, 80%, 90%, 95%, 98%, and 99%) identical to SEQ ID NO: 1. In certainembodiments, the “Cas mutant” or “Cas variant” binds to an RNA molecule(e.g., a sgRNA). In certain embodiments, the “Cas mutant” or “Casvariant” is targeted to a specific polynucleotide sequence via the RNAmolecule.

Fusion Proteins

In certain embodiments, the Cas protein is fused to another protein orpolypeptide heterologous to the Cas protein to create a fusion protein.In certain embodiments, the heterologous sequence includes one or moreeffector domains, such as a cleavage domain, a transcriptionalactivation domain, a transcriptional repressor domain, or an epigeneticmodification domain. Additional examples of the effector domain includea nuclear localization signal, cell-penetrating of translocation domain,or a marker domain. In certain embodiments, the effector domain islocated at the N-terminal, the C-terminal, or in an internal location ofthe fusion protein. In certain embodiments, the Cas protein of thefusion protein is or is derived from a Cas9 protein. In certainembodiments, the Cas protein of the fusion protein is or is derived froma modified or mutated Cas protein in which all the nuclease domains havebeen inactivated or deleted. In certain embodiments, the Cas protein ofthe fusion protein is or is derived from a modified or mutated Casprotein that lacks nuclease activity. In certain embodiments, the RuvCand/or HNH domains of the Cas protein are modified or mutated such thatthey no longer possess nuclease activity.

Cleavage Domains

In certain embodiments, the effector domain of the fusion protein is acleavage domain. As used herein, a “cleavage domain” refers to a domainthat cleaves DNA. The cleavage domain can be obtained from anyendonuclease or exonuclease. Non-limiting examples of endonucleases fromwhich a cleavage domain can be derived include restriction endonucleasesand homing endonucleases. See, for example, New England Biolabs Catalogor Belfort et al. (1997) Nucleic Acids Res. 25, 3379-88. Additionalenzymes that cleave DNA are known (e.g., 51 Nuclease; mung beannuclease; pancreatic DNase I; micrococcal nuclease; yeast HOendonuclease). See also Linn et al. (eds.) “Nucleases,” Cold SpringHarbor Laboratory Press, 1993. One or more of these enzymes (orfunctional fragments thereof) can be used as a source of cleavagedomains.

In certain embodiments, the cleavage domain can be derived from a typeII-S endonuclease. Type II-S endonucleases cleave DNA specifically atsites that are typically several base pairs away from the DNArecognition site of the endonuclease and, as such, have separablerecognition and cleavage domains. These enzymes generally arc monomersthat transiently associate to form dimers to cleave each strand of DNAat staggered locations. Non-limiting examples of suitable type II-Sendonucleases include BfiI, BpmI, BsaI, BsgI, BsmBI, BsmI, BspMI, FokI,MbolI, and SapI. In certain embodiments, the cleavage domain of thefusion protein is a FokI cleavage domain or a fragment or derivativethereof. See Miller et al. (2007) Nat. Biotechnol. 25, 778-85; Szezpeket al. (2007) Nat. Biotechnol. 25, 786-93; Doyon et al. (2011) Nat.Methods, 8, 74-81.

Transcriptional Activation Domains

In certain embodiments, the effector domain of the fusion protein is atranscriptional activation domain. In general, a transcriptionalactivation domain interacts with transcriptional control elements and/ortranscriptional regulatory proteins (i.e., transcription factors, RNApolymerases, etc.) to increase and/or activate transcription of a gene.In certain embodiments, the transcriptional activation domain is aherpes simplex virus VP16 activation domain, VP64 (which is a tetramericderivative of VP16), a NFκB p65 activation domain, p53 activationdomains 1 and 2, a CREB (cAMP response element binding protein)activation domain, an E2A activation domain, or an NFAT (nuclear factorof activated T-cells) activation domain. In certain embodiments, thetranscriptional activation domain is Gal4, Gen4, MLL, Rtg3, Gln3, Oaf1,Pip2, Pdr1, Pdr3, Pho4, or Leu3. The transcriptional activation domainmay be wild type, or it may be a modified or truncated version of theoriginal transcriptional activation domain.

Transcriptional Repressor Domains

In certain embodiments, the effector domain of the fusion protein is atranscriptional repressor domain. In general, a transcriptionalrepressor domain interacts with transcriptional control elements and/ortranscriptional regulatory proteins (i.e., transcription factors, RNApolymerases, etc.) to decrease and/or prohibit transcription of a gene.In certain embodiments, the transcriptional repressor domains isinducible cAMP early repressor (ICER) domains, Kruppel-associated box A(KRAB-A) repressor domains, YY1 glycine rich repressor domains, Sp1-likerepressors, E(spI) repressors, IκB repressor, or MeCP2.

Epigenetic Modification Domains

In certain embodiments, the effector domain of the fusion protein is anepigenetic modification domain. In general, epigenetic modificationdomains alter gene expression by modifying the histone structure and/orchromosomal structure. In certain embodiments, the epigeneticmodification domains is a histone acetyl transferase domain, a histonedeacetylase domain, a histone methyltransferase domain, a histonedemethylase domain, a DNA methyltransferase domain, or a DNA demethylasedomain.

Additional Domains

In certain embodiments, the fusion protein further comprises at leastone additional domain. Non-limiting examples of suitable additionaldomains include nuclear localization signals (NLSs), cell-penetrating ortranslocation domains, and marker domains. An NLS generally comprises astretch of basic amino acids. See, e.g., Lange et al. (2007) J. BiolChem., 282, 5101-5. For example, in certain embodiments, the NLS is amonopartite sequence, such as PKKKRKV (SEQ ID NO: 2) or PKKKRRV (SEQ IDNO: 3). In certain embodiments, the NLS is a bipartite sequence. Incertain embodiments, the NLS is KRPAATKKAGQAKKKK (SEQ ID NO: 4).

In certain embodiments, the fusion protein comprises at least onecell-penetrating domain. In certain embodiments, the cell-penetratingdomain is a cell-penetrating peptide sequence derived from the HIV-1 TATprotein. As an example, the TAT cell-penetrating sequence can beGRKKRRQRRRPPQPKKKRKV (SEQ ID NO: 5). In certain embodiments, thecell-penetrating domain is TLM (PLSSBFSRIGDPPKKKRKV; SEQ ID NO: 6), acell-penetrating peptide sequence derived from the human hepatitis Bvirus. In certain embodiments, the cell-penetrating domain is MPG(GALFLGWLGAAGSTMGAPKKKRKV; SEQ ID NO: 7 or GALFLGFLGAAGSTMGAWSQPKKKRKV;SEQ ID NO: 8). In certain embodiments, the cell-penetrating domain isPcp-1 (KETWWETWWTEWSQPKKKRKV; SEQ ID NO: 9), VP22, a cell penetratingpeptide from Herpes simplex virus, or a polyarginine peptide sequence.

In certain embodiments, the fusion protein comprises at least one markerdomain. Non-limiting examples of marker domains include fluorescentproteins, purification tags, and epitope tags. In certain embodiments,the marker domain is a fluorescent protein. Non limiting examples ofsuitable fluorescent proteins include green fluorescent proteins (e.g.,GFP, GFP-2, tagGFP, turbpGFP, EGFP, Emerald, Azami Green, MonomelicAzami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins(e.g. YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), bluefluorescent proteins (e.g. EBFP, EBFP2, Azurite, mKalama1, GFPuv,Sapphire, T-sapphire,), cyan fluorescent proteins )e.g. ECFP, Cerulean,CyPct, AmCyan1, Midoriishi-Cyan), red fluorescent proteins (mKate,mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRcd-Express, DsRed2,DsRed-Monomer, HcRed-Tandem, HcRc1, AsRed2, eqFP611, mRasberry,mStrawbeny, Jred), orange fluorescent proteins (mOrangc, mKO,Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato) andany other suitable fluorescent protein. In certain embodiments, themarker domain is a purification tag and/or an epitope tag. Exemplarytags include, but are not limited to, glutathione-S-transferase (GST),chitin binding protein (CBP), maltose binding protein, thioredoxin(TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5,AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP,Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6xHis, biotin carboxyl carrierprotein (BCCP), and calmodulin.

V. Uses and Methods

In one aspect, the present invention provides a method for cleaving atarget polynucleotide with a Cas protein. The method comprisescontacting the target polynucleotide with (i) a guide RNA or a set ofguide RNA molecules described herein, and (ii) a Cas protein. In certainembodiments, the method results in a double-strand break in the targetpolynucleotide. In certain embodiments, the Cas protein is a Cas proteinhaving a single-strand nicking activity. In certain embodiments, themethod results in a single-strand break in the target polynucleotide. Incertain embodiments, a complex comprising a guide RNA and Cas proteinhaving a single-strand nicking activity is used for sequence-targetedsingle-stranded DNA cleavage, i.e., nicking.

In one aspect, the present invention provides a method for cleaving twoor more target polynucleotides with a Cas protein. The method comprisescontacting the target polynucleotides with (i) a set of guide RNAmolecules described herein, and (ii) a Cas protein. In certainembodiments, the method results in double-strand breaks in the targetpolynucleotides. In certain embodimerits, the Cas protein is a Casprotein having a single-strand nicking activity. In certain embodiments,the method results in single-strand breaks in the targetpolynucleotides. In certain embodiments, a complex comprising a guideRNA and Cas protein having a single-strand nicking activity is used forsequence-targeted single-stranded DNA cleavage, i.e., nicking.

In one aspect, the present invention provides a method for binding atarget polynucleotide with a Cas protein. The method comprisescontacting the target polynucleotide with (i) a guide RNA or a set ofguide RNA molecules described herein and (ii) a Cas protein, to resultin binding of the target polynucleotide with the Cas protein. In certainembodiments, the Cas protein-is a Cas variant. In certain embodiments,the Cas variant lacks some or all nuclease activity relative to acounterpart wild-type Cas protein.

In one aspect, the present invention provides a method for binding twoor more target polynucleotides with a Cas protein. The method comprisescontacting the target polynucleotides with (i) a set of RNA moleculesdescribed herein and (ii) a Cas protein, to result in binding of thetarget polynucleotides with the Cas protein. In certain embodiments, theCas protein is a Cas variant In certain embodiments, the Cas variantlacks some or all nuclease activity relative to a counterpart wild-typeCas protein.

In one aspect, the present invention provides a method for targeting aCas protein to a target polynucleotide. The method comprises contactingthe Cas protein with a guide RNA or a set of guide RNA moleculesdescribed herein. In certain embodiments, the method results informationof a guide RNA:Cas protein complex. In certain embodiments, the Casprotein is a wild type Cas9 protein. In certain embodiments, the Casprotein is a mutant or variant of a Cas9 protein. In certainembodiments, the Cas protein is a Cas protein having a single-strandnicking activity. In certain embodiments, the Cas protein is a Casprotein lacking nuclease activity (e.g., a nuclease-deficient mutant ofCas protein). In certain embodiments, the Cas protein is part of afusion protein (e.g., a fusion protein comprising (i) the Cas proteinand (ii) a heterologous polypeptide).

In one aspect, the present invention provides a method for targeting aCas protein to two or more target polynucleotides. The method comprisescontacting the Cas protein with a set of guide RNA molecules describedherein. In certain embodiments, the method results in formation of aguide RNA:Cas protein complex. In certain embodiments, the Cas proteinis a wild type Cas9 protein. In certain embodiments, the Cas protein isa mutant or variant of a Cas9 protein. In certain embodiments, the Casprotein is a Cas protein having a single-strand nicking activity. Incertain embodiments, the Cas protein is a Cas protein lacking nucleaseactivity (e.g., a nuclease-deficient mutant of Cas protein). In certainembodiments, the Cas protein is part of a fusion protein (e.g., a fusionprotein comprising (i) the Cas protein or and (ii) a heterologouspolypeptide).

In certain embodiments, the guide RNA is introduced into a cell bytransfection. Techniques for RNA transfection arc known in the art andinclude electroporation and lipofection. Effective techniques for RNAtransfection depend mostly on cell type. See, e.g., Lujambio et al.(Spanish National Cancer Centre) Cancer Res. February 2007, whichdescribes transfection of HTC-116 colon cancer cells and usesOligofectamine (Invitrogen) for transfection of commercially obtained,modified miRNA or precursor miRNA. See also, Cho et al. (Seoul NationalUniv.) Nat. Biotechnol. March 2013, which describes transfection of K562cells and uses 4D Nucleofection™ (Lonza) electroporation fortransfection of transcribed sgRNAs (about 60 nts long). Techniques fortransfection of RNA are also known in the art. For example, therapeuticRNA has been delivered in non-pathogenic E. coli coated with Invasinprotein (to facilitate uptake into cells expressing β-1 integrinprotein) and with the E. coli encoded to express lysteriolysin Opore-forming protein to permit the shRNA to pass from the E. coli intothe cytoplasm. Sec also Cho el al. (Seoul National Univ.) Nat.Biotechnol. March 2013.

In certain embodiments, the guide RNA is introduced or delivered intocells. Technologies that can be used for delivery of guide RNA includethose that utilize encapsulation by biodegradable polymers, liposomes,or nanoparticles. Such polymers, liposomes, and nanoparticles can bedelivered intravenously. In certain embodiments, for in vivo delivery,guide RNA can be injected into a tissue site or administeredsystemically. In vivo delivery can also be effected by a beta-glucandelivery system, such as those described in U.S. Pat. Nos. 5.032,401 and5,607,677, and U.S. Publication No. 2005/0281781, which are herebyincorporated by reference in their entirety. In certain embodiments,guide RNA or a delivery vehicle containing guide RNA is targeted to aparticular tissue or body compartment. For example, in certainembodiments, to target exogenous RNA to other tissues, syntheticcarriers are decorated with cell-specific ligands or aptamers forreceptor uptake, e.g., RNA encased in cyclodextrin nanoparticles coatedwith PEG and functionalized with human transferrin protein for uptakevia the transferrin receptor which is highly expressed in tumor cells.Further approaches are described herein below or known in the art

The present invention has been tested in human cells as described inHendel et al., Nat. Biotechnol. (2015) 33:9, 985-9 (which isincorporated in this application in its entirety). In the cited work,modified guide RNA was introduced into K562 cells, human primary Tcells, and CD34+ hematopoietic stem and progenitor cells (HSPCs). Themodified guide RNA significantly enhanced genome editing efficiencies inhuman cells, including human primary T cells and CD34+ HSPCs as comparedto unmodified guide RNA.

FIGS. 11A and 11B illustrate experimental results showing that genedisruption in human cell lines can be achieved by high frequencies ofindels or by cleavage-stimulated homologous recombination usingsynthesized and chemically modified sgRNAs disclosed herein. Genedisruption by mutagenic NHEJ was measured by deep sequencing of PGRamplicons (FIG. 12A) or gene addition by HR at the three loci IL2RG, HBBand CCR5 in K562 cells induced by Cas9 in combination with, syntheticsgRNAs (FIG. 12B). The synthetic sgRNAs were delivered at 1 μg (lightshade) or 20 μg (dark shade) per 1 million cells. Cas9 was expressedfrom a plasmid (2 μg) and for HR experiments 5 μg of GFP-encoding donorplasmid was included. As a positive control, 2 μg of sgRNA plasmidencoding both the sgRNA and the Cas9 protein was used (gray bars). Barsrepresent average values+s.e.m., n=3.

FIGS. 12A, 12B, 12C and 12D illustrate experimental results showing thatchemically modified sgRNAs as described herein can be used to achievehigh frequencies of gene disruption or targeted genome editing instimulated primary human T cells and CD34+ hematopoietic stem andprogenitor cells (HSPCs).

FIG. 12A illustrates results from primary human T cells nucleofectedwith 10 μg of a synthetic CCR5 sgRNAs and either 15 μg Cas9 mRNA or 1 μgCas9-encoding plasmid. 1 μg sgRNA plasmid encoding both the sgRNA andCas9 protein was included for comparison. The bars represent averageindel frequencies for three different donors+s.e.m., n=6, as measured byTIDE (tracking of indels by decomposition) analysis of PCR ampliconsspanning the sgRNA target site, and using a mock-treated sample ascontrol reference. Delivery of Cas9 mRNA with the unmodified or theM-modified sgRNA, and nucleofection of the plasmid encoding both thesgRNA and Cas9, did not give rise to allele modification frequenciesabove background. Co-transfection of the MSP-modified sgRNA with DNAexpression plasmid for Cas9 generated 9.3% indel frequency. Cas9 mRNAwith either the MS- or MSP-modified sgRNA generated 48.7% and 47.9%indel frequencies, respectively.

FIG. 12B illustrates results from stimulated T cells. The cells werenucleofected as above, but with 15 μg Cas9 protein complexed with a 2.5molar excess of the indicated synthetic CCR5 sgRNAs. Indel frequencieswere measured by TIDE analysis. The bars represent average indelfrequencies for three different donors+s.e.m., n=6. A 2.4-foldimprovement in indel frequencies of the MS-modified sgRNA over theunmodified sgRNA (30.7% vs. 12.8%) was observed for chemically modifiedsgRNAs when delivered complexed with Cas9 protein. These resultsestablish that chemically modified sgRNAs can be used for genome editingof stimulated T cells when delivered complexed with Cas9 protein.

FIG. 12C illustrates results from human peripheral blood CD34+ HSPCs.500,000 mobilized cells were nucleofected with 10 μg of the indicatedsynthetic sgRNAs targeting IL2RG or HBB and either 15 μg Cas9 mRNA or 1μg Cas9 plasmid. 1 μg of sgRNA plasmid encoding both the sgRNA and Cas9protein was included for comparison. Bars represent average indelfrequencies+s.e.m., n=3, as measured by T7 endonuclease cleavage assay.Indels were not detected at either locus using the unmodified orM-modified sgRNAs when co-transfected with Cas9 mRNA. However, the IL2RGMS- and MSP-modified sgRNAs showed 17.5% and 17.7% indel frequencies,respectively, and 23.4% and 22.0%, respectively, for the HBB MS- andMSP-modified sgRNAs.

FIG. 12D illustrates results from stimulated T cells or mobilized humanperipheral blood CD34+ HSPCs. One million cells were nucleofected with15 μg Cas9 mRNA and 10 μg of the indicated synthetic CCR5 sgRNAs. Arecent study showed that the simultaneous use of two sgRNAs couldimprove gene disruption in human primary T cells and in CD34+ HSPCs.See, e.g., Mandal et al. (2014) Cell Stem Cell, 15, 643-52. MS- andMSP-modified CCR5 sgRNAs were chemically synthesized with the sequencesreported in Mandal study (termed ‘D’ and ‘Q’), which cut 205 base pairsapart. When used in combination, the amount of each sgRNA was 5 μg.Indel frequencies for samples with single sgRNAs were measured by TIDEanalysis as above and allele disruption frequencies for samples with twosgRNAs were measured by sequencing of cloned PCR products. The barsrepresent average indel frequencies+s.e.m., n=3. In T cells, the ‘D’sgRNA alone gave rise to 56.0% and 56.3% indels for the MS- andMSP-modified sgRNA, respectively, and the ‘Q’ sgRNA gave rise to 62.6%and 69.6% indels, respectively. When used in combination, thefrequencies of allele modification increased, as we observed 73.9% and93.1% indels for the MS- and MSP-modified sgRNAs, respectively, of whichthe majority of the modification events were deletions between the twosgRNA target sites. In CD34+ HSPCs, observations were similar though theoverall frequencies were lower. For the ‘D’ sgRNA, allele modificationfrequencies of 9.8% and 11.2% were observed for the MS- and MSP-modifiedsgRNA, respectively, and 17.8% and 19.2% for the ‘Q’ sgRNA. When used incombination the frequencies increased to 37.8% and 43.0% for the MS- andMSP-modified sgRNAs, respectively This shows that the use of twochemically modified sgRNAs is a highly effective way to facilitate genedisruption in primary human T cells and CD34+ HSPCs.

Examples of other uses include genomic editing and gene expressionregulation as described below.

Genomic Editing

In one aspect, the present invention provides a method for genomicediting to modify a DNA sequence in vivo or in vitro (“in vitro”includes, without being limited to, a cell-free system, a cell lysate,an isolated component of a cell, and a cell outside of a livingorganism). The DNA sequence may comprise a chromosomal sequence, anepisomal sequence, a plasmid, a mitochondrial DNA sequence, or afunctional intergenic sequence, such, as an enhancer sequence or a DNAsequence for a non-coding RNA. The method comprises contacting the DNAsequence with (i) a guide RNA or a set of guide RNA molecules describedherein, and (ii) a Cas protein. In certain embodiments, the DNA sequenceis contacted outside of a cell. In certain embodiments, the DNA sequenceis located in the genome within a cell and is contacted in vitro or invivo. In certain embodiments, the cell is within an organism or tissue.In certain embodiments, the cell is a human cell, a non-human mammaliancell, a stem cell, a non-mammalian vertebrate cell, an invertebratecell, a plant cell, a single cell organism, or an embryo. In certainembodiments, the guide RNA aids in targeting the Cas protein to atargeted site in the DNA sequence. In certain embodiments, the Casprotein cleaves at least one strand of the DNA sequence at the targetedsite. In certain embodiments, the Cas protein cleaves both strands ofthe DNA sequence at the targeted site.

In certain embodiments, the method further comprises introducing the Casprotein into a cell or another system. In certain embodiments, the Casprotein is introduced as a purified or non-purified protein. In certainembodiments, the Cas protein is introduced via an mRNA encoding the Casprotein. In certain embodiments, the Cas protein is introduced via alinear or circular DNA encoding the Cas protein. In certain embodiments,the cell or system comprises a Cas protein or a nucleic acid encoding aCas protein.

In certain embodiments, a double-stranded break can be repaired via anerror-prone, non-homologous end-joining (“NHEJ”) repair process. Incertain embodiments, a double-stranded break can be repaired by ahomology-directed repair (HDR) process such that a donor sequence in adonor polynucleotide can be integrated into or exchanged with thetargeted DNA sequence.

In certain embodiments, the method further comprises introducing atleast one donor polynucleotide into the cell or system. In certainembodiments, the donor polynucleotide comprises at least one homologoussequence having substantial sequence identity with a sequence, on eitherside of the targeted site in the DNA sequence. In certain embodiments,the donor polynucleotide comprises a donor sequence that can beintegrated into or exchanged with the DNA sequence via homology-directedrepair, such as homologous recombination.

In certain embodiments, the donor polynucleotide includes an upstreamhomologous sequence and a downstream homologous sequence, each of whichhave substantial sequence identity to sequences located upstream anddownstream, respectively, of the targeted site in the DNA sequence.These sequence similarities permit, for example, homologousrecombination between the donor polynucleotide and the targeted DNAsequence such that the donor sequence can be integrated into (orexchanged with) the DNA sequence targeted.

In certain embodiments, the target site(s) in the DNA sequence spans oris adjacent to a mutation, e.g., point mutation, a translocation or aninversion which may cause or be associated with a disorder. In certainembodiments, the method comprises correcting the mutation by introducinginto the cell or system at least one donor polynucleotide comprising (i)a wild type counterpart of the mutation and (ii) at least one homologoussequence having substantial sequence identity with a sequence on oneside of the targeted site in the DNA sequence. In certain embodiments,the donor polynucleotide comprises a homologous sequence havingsubstantial sequence identity with a sequence on both sides of thetargeted site in the DNA sequence.

In certain embodiments, the donor polynucleotide comprises an exogenoussequence that can be integrated into or exchanged with the targeted DNAsequence via a homology-directed repair process, such as homologousrecombination. In certain embodiments, the exogenous sequence comprisesa protein coding gene, which, optionally, is operably linked to anexogenous promoter control sequence. Thus, in certain embodiments, uponintegration of the exogenous sequence, a cell can express a proteinencoded by the integrated gene. In certain embodiments, the exogenoussequence is integrated into the targeted DNA sequence such that itsexpression in the recipient cell or system is regulated by the exogenouspromoter control sequence. Integration of an exogenous gene into thetargeted DNA sequence is termed a “knock in.” In other embodiments, theexogenous sequence can be a transcriptional control sequence, anotherexpression control sequence, an RNA coding sequence, and the like.

In certain embodiments, the donor polynucleotide comprises a sequencethat is essentially identical to a portion of the DNA sequence at ornear the targeted site, but comprises at least one nucleotide change.For example, in certain embodiments, the donor sequence comprises amodified or mutated version of the DNA sequence at or near the targetedsite such that, upon integration or exchange with the targeted site, theresulting sequence at the targeted site comprises at least onenucleotide change. In certain embodiments, the at least one nucleotidechange is an insertion of one or more nucleotides, a deletion of one ormore nucleotides, a substitution of one or more nucleotides, orcombinations thereof. As a consequence of the integration of themodified sequence, the cell may produce a modified gene product from thetargeted DNA sequence.

In certain embodiments, the methods are for multiplex applications. Incertain embodiments, the methods comprise introducing a library of guideRNAs into the cell or system. In certain embodiments, the librarycomprises at least 100 unique guide sequences. In certain embodiments,the library comprises at least 1,000 unique guide sequences. In certainembodiments, the library comprises at least 10,000 unique guidesequences. In certain embodiments, the library comprises at least100,000 unique guide sequences. In certain embodiments, the librarycomprises at least 1,000,000 unique guide sequences. In certainembodiments, the library targets at least 10 different polynucleotidesor at least 10 different sequences within one or more polynucleotides.In certain embodiments, the library targets at least 100 differentpolynucleotides or at least 100 different sequences within one or morepolynucleotides. In certain embodiments, the library targets at least1,000 different polynucleotides or at least 1,000 different sequenceswithin one or more polynucleotides. In certain embodiments, the librarytargets at least 10,000 different polynucleotides or at least 10,000different sequences within one or more polynucleotides. In certainembodiments, the library targets at least 100,000 differentpolynucleotides or at least 100,000 different sequences within one ormore polynucleotides. In certain embodiments, the library targets atleast 1,000,000 different polynucleotides or at least 1,000,000different sequences within one or more polynucleotides.

Genomic Editing in Human and Mammalian Cells

Embodiments of the present invention are useful in methods for genomicediting to modify a target polynucleotide, for example a DNA sequence;in a mammalian cell.

In certain embodiments, the DNA sequence is a chromosomal sequence. Incertain embodiments, the DNA sequence is a protein-coding sequence. Incertain embodiments, the DNA sequence is a functional intergenicsequence, such as an enhancer sequence or a non-coding sequence. Incertain embodiments, the DNA is part of a human gene. In some suchembodiments, the human gene is the clathrin light chain (CLTA1) gene,the human interleukin 2 receptor gamma (IL2RG) gene, the human cytotoxicT-lymphocyte-associated protein 4 (CLTA4) gene, the human protocadherinalpha 4 (PCDHA4) gene, the human engrailed homeobox 1 (EN1) gene), thehuman hemoglobin beta (HBB) gene, which can harbor mutations responsiblefor sickle cell anemia and thalassemias, or the human chemokine (C-Cmotif) receptor 5 (CCR5) gene which encodes a co-receptor of HIV.

In certain embodiments, the mammalian cell is a human cell. In some suchembodiments, the human cell is a primary human cell. In furtherembodiments, the primary human cell is a human primary T cell. The humanprimary T cell may be stimulated or unstimulated. In certainembodiments, the human cell is a stem/progenitor cell, such as a CD34+hematopoietic stem and progenitor cell (HSPC). In certain embodiments,the human cell is from a cultured cell line, for example such as can beobtained commercially. Exemplary cell lines include K562 cells, a humanmyelogenous leukemia line.

In certain embodiments, the cell is within a living organism. In certainother embodiments, the cell is outside of a living organism.

The method comprises contacting the DNA sequence with (i) a guide RNA ora set of guide RNA molecules described herein, and (ii) a Cas protein.

In certain embodiments, the method further comprises introducing ordelivering the guide RNA into the cell. In some such embodiments, theguide RNA is introduced into a cell by transfection. Techniques for RNAtransfection are known in the art and include electroporation andlipofection. In other embodiments, the guide RNA is introduced into acell (and, more particularly, a cell nucleus) by nucleofection.Techniques for nucleofection are known in the art and may utilizenucleofection devices such as the LonzaNucleofector 2b or the Lonza4D-Nucleofector and associated reagents.

In certain embodiments, the method further comprises introducing ordelivering the Cas protein into the cell. In some such embodiments, theCas protein is introduced as a purified or non-purified protein. Inother embodiments, the Cas protein is introduced via an mRNA encodingthe Cas protein. In some such embodiments, the mRNA encoding the Casprotein is introduced into the cell by transfection. In otherembodiments, the mRNA encoding the Cas protein is introduced into a cell(and, more particularly, a cell nucleus) by nucleofection.

In certain embodiments, the method employs ribonucleoprotein (RNP)-baseddelivery such that the Cas protein is introduced into the cell in acomplex with the guide RNA. For example, a Cas9 protein may be complexedwith a guide RNA in a Cas9:gRNA complex, which allows for co-delivery ofthe gRNA and Cas protein. For example, the Cas:gRNA complex may benucleofected into cells.

In certain embodiments, the method employs an all-RNA delivery platform.For example, in some such embodiments, the guide RNA and the mRNAencoding the Cas protein are introduced into the cell simultaneously orsubstantially simultaneously (e.g., by co-transfection orco-nucleofection). In certain embodiments, co-delivery of Cas mRNA andmodified gRNA results in higher editing frequencies as compared toco-delivery of Cas mRNA and unmodified gRNA. In particular, gRNA having2′-O-methyl-3′-phosphorothioate (MS), or 2′-O-methyl-3′-thioPACE (MSP)incorporated at three terminal nucleotides at both the 5′ and 3′ ends,provide higher editing frequencies as compared to unmodified gRNA.

In certain embodiments, the guide RNA and the mRNA encoding the Casprotein are introduced into the cell sequentially; that is, the guideRNA and the mRNA encoding the Cas protein are introduced into the cellat different times. The time period between the introduction of eachagent may range from a few minutes (or less) to several hours or days.For example, in some such embodiments, gRNA is delivered first, followedby delivery of Cas mRNA 4, 8, 12 or 24 hours later. In other suchembodiments, Cas mRNA is delivered first, followed by delivery of gRNA4, 8, 12 or 24 hours later. In some particular embodiments, delivery ofmodified gRNA first, followed by delivery of Cas mRNA results in Higherediting frequencies as compared to delivery of unmodified gRNA followedby delivery of Cas mRNA.

In certain embodiments, the gRNA is introduced into the cell togetherwith a DNA plasmid encoding the Cas protein. In some such embodiments,the gRNA and the DNA plasmid encoding the Cas protein are introducedinto the cell by nucleofection. In some particular embodiments, anRNP-based delivery platform or an all-RNA delivery platform provideslower cytotoxicity in primary cells than a DNA plasmid-based deliverysystem.

In certain embodiments, the method provides significantly enhancedgenome editing efficiencies in human cells, including human primary Tcells and CD34+ HSPCs.

In certain embodiments, modified gRNA increases the frequency ofinsertions or deletions (indels), which may be indicative of mutagenicNHEJ and gene disruption, relative to unmodified gRNA. In particular,modified gRNA having 2′-O-methyl-3′-phosphorothioate (MS) or2′-O-methyl-3′-thioPAGE (MSP) incorporated at three terminal nucleotidesat both the 5′ and 3′ ends, increases the frequency of indels relativeto unmodified gRNA.

In certain embodiments, co-delivery of modified gRNA and Cas mRNA tohuman primary T cells increases the frequency of indels as compared toco-delivery of unmodified gRNA and Cas mRNA. In particular, modifiedgRNA having 2′-O-methyl-3′-phosphorothioate (MS) or2-O-methyl-3′-thioPACE (MSP) incorporated at three terminal nucleotidesat both the 5′ and 3′ ends, increases the frequency of indels in humanprimary T cells relative to unmodified gRNA.

In certain embodiments, modified gRNA improves gRNA stability relativeto unmodified gRNA. As one example, gRNA having 2′-O-methyl (M)incorporated at three terminal nucleotides at both the 5′ and 3′ ends,modestly improves stability against nucleases and also improves basepairing thermostability over unmodified gRNA. As another example, gRNAhaving 2′-O-methyl-3′-phosphorothioate (MS) or 2′-O-methyl-3′-thioPACE(MSP) incorporated at three terminal nucleotides at both the 5′ and 3′ends, dramatically improves stability against nucleases relative tounmodified gRNA. It is contemplated that gRNA end modifications enhanceintracellular stability against exonucleases, thus enabling increasedefficacy of genome editing when Cas mRNA and gRNA are co-delivered orsequentially delivered into human cells.

In certain embodiments, modified gRNA stimulates gene targeting, which,in turn, allows for gene editing by, for example, homologousrecombination or NHEJ. In particular, gRNA having2′-O-methyl-3′-phosphorothioate (MS), or 2-O-methyl-3′-thioPACE (MSP)incorporated at three terminal nucleotides at both the 5′ and 3′ ends,stimulates higher levels of homologous recombination than unmodifiedgRNA.

In certain embodiments, modified gRNA retains high specificity. Incertain embodiments, the ratio of on-target to off-target indelfrequencies is improved with modified gRNA as compared to unmodifiedgRNA. In certain embodiments, modified gRNA delivered in an RNP complexwith a Cas protein provides significantly better on-target:off-targetratios compared to a DNA plasmid-based delivery system.

Gene Expression Regulation

In certain embodiments, the guide RNA described herein is used forregulating transcription or expression of a gene of interest. Forexample, in certain embodiments, a fusion protein comprising a Casprotein (e.g., a nuclease-deficient Cas9) and a transcription activatorpolypeptide is used to increase transcription of a gene. Similarly, incertain embodiments, a fusion protein comprising a Cas protein (e.g., anuclease-deficient Cas9) and a repressor polypeptide is used toknock-down gene expression by interfering with transcription of thegene.

In at least one aspect, the present invention provides a method forregulating the expression of a gene of interest in vivo or in vitro. Themethod comprises introducing into a cell or another system(i) asynthetic guide RNA described herein, and (ii) a fusion protein. Incertain embodiments, the fusion protein comprises a Cas protein and aneffector domain, such as a transcriptional activation domain, atranscriptional repressor domain, or an epigenetic modification domain.In certain embodiments, the fusion protein comprises a mutated Casprotein, such as a Cas9 protein that is a null nuclease. In certainembodiments, the Cas protein contains one or more mutations, such asD10A, H840A and/or N863A.

In certain embodiments, the fusion protein is introduced into the cellor system as a purified or non-purified protein. In certain embodiments,the fusion protein is introduced into the cell or system via an mRNAencoding the fusion protein. In certain embodiments, the fusion proteinis introduced into the cell or system via a linear or circular DNAencoding the fusion protein,

In certain embodiments, the guide RNA aids in directing the fusionprotein to a specific target polynucleotide comprising a chromosomalsequence, an episomal sequence, a plasmid, a mitochondrial DNA sequence,or a functional intergenic sequence, such as an enhancer or the DNAsequence for a non-coding RNA. In certain embodiments, the effectordomain regulates expression of a sequence in the target polynucleotide.A guide RNA for modulating gene expression can be designed to target anydesired endogenous gene or sequence encoding a functional RNA. A genomictarget sequence can be selected in proximity of the transcription startsite of the endogenous gene, or alternatively, in proximity of thetranslation initiation site of the endogenous gene. In certainembodiments, the target sequence is in a region of the DNA that istraditionally termed the promoter proximal region of a gene. In certainembodiments, the target sequence lies in a region from about 1,000 basepairs upstream of the transcription start site to about 1,000 base pairsdownstream of the transcription start site. In certain embodiments, thetarget sequence is remote from the start site for transcription of thegene (e.g., on another chromosome).

In certain embodiments, the methods are for multiplex applications. Incertain embodiments, the methods comprise introducing a library of guideRNAs into the cell or system. In certain embodiments, the librarycomprises at least 100, at least 1,000, at least 10,000, at least100,000, or at least 1,000,000 unique guide sequences. In certainembodiments, the library targets at least 10 different polynucleotidesor at least 10 different sequences within one or more polynucleotides.In certain embodiments, the library targets at least 100 differentpolynucleotides or at least 100 different sequences within one or morepolynucleotides. In certain embodiments, the library targets at least1,000 different polynucleotides or at least 1,000 different sequenceswithin one or more polynucleotides. In certain embodiments, the librarytargets at least 10,000 different polynucleotides or at least 10,000different sequences within one or more polynucleotides. In certainembodiments, the library targets at least 100,000 differentpolynucleotides or at least 100,000 different sequences within one ormore polynucleotides. In certain embodiments, the library targets atleast 1,000,000 different polynucleotides or at least 1,000,000different sequences within one or more polynucleotides.

Kits

In one aspect, the present invention provides kits containing reagentsfor performing the above-described methods, including producing gRNA:Casprotein complex and/or supporting its activity for binding, nicking orcleaving target polynucleotide. In certain embodiments, one or more ofthe reaction components, e.g., one or more guide RNAs and Cas proteins,for the methods disclosed herein, can be supplied in the form of a kitfor use. In certain embodiments, the kit comprises a Cas protein or anucleic acid encoding the Cas protein, and one or more guide RNAsdescribed herein or a set or library of guide RNAs. In certainembodiments, the kit includes one or more other reaction components. Incertain embodiments, an appropriate amount of one or more reactioncomponents is provided in one or more containers or held on a substrate.

Examples of additional components of the kits include; but are notlimited to, one or more different polymerases, one or more host cells,one or more reagents for introducing foreign nucleic acid into hostcells, one or more reagents (e.g., probes or PCR primers) for detectingexpression of the guide RNA and/or the Cas mRNA or protein or forverifying the status of the target nucleic acid, and buffers,transfection reagents or culture media for the reactions (in 1× or moreconcentrated forms). In certain embodiments, the kit includes one ormore of the following components: biochemical and physical supports;terminating, modifying and/or digesting reagents; osmolytes; andapparati for reaction, transfection and/or detection.

The reaction components used can be provided in a variety of forms. Forexample, the components (e.g., enzymes, RNAs, probes and/or primers) canbe suspended in an aqueous solution or bound to a bead or as afreeze-dried or lyophilized powder or pellet. In the latter case, thecomponents, when reconstituted, form a complete mixture of componentsfor use in an assay. The kits of the invention can be provided at anysuitable temperature. For example, for storage of kits containingprotein components or complexes thereof in a liquid, it is preferredthat they are provided and maintained below 0° C., preferably at about−20° C., possibly in a freeze-resistant solution containing glycerol orother suitable antifreeze.

A kit or system may contain, in an amount sufficient for at least oneassay, any combination of the components described herein. In someapplications, one or more reaction components may be provided inpre-measured single use amounts in individual, typically disposable,tubes or equivalent containers. With such an arrangement, a RNA-guidednuclease reaction can be performed by adding a target nucleic acid, or asample or cell containing the target nucleic acid, to the individualtubes directly. The amount of a component supplied in the kit can be anyappropriate amount and may depend on the market to which the product isdirected. The container(s) in which the components are supplied can beany conventional container that is capable of holding the supplied form,for instance, microfuge tubes, microtiter plates, ampoules, bottles, orintegral testing devices, such as fluidic devices, cartridges, lateralflow, or other similar devices.

The kits can also include packaging materials for holding the containeror combination of containers. Typical packaging materials for such kitsand systems include solid matrices (e.g., glass, plastic, paper, foil,micro-particles and the like) that hold the reaction components ordetection probes in any of a variety Of configurations (e.g., in a vial,microtiter plate well, microarray, and the like). The kits may furtherinclude instructions recorded in a tangible form for use of thecomponents.

EXAMPLES Example 1

To evaluate the ability of the chemically synthesized guide RNAs totarget and cleave a DNA target sequence, an in vitro cleavage assay wasdeveloped. Briefly, as shown in FIG. 3, ˜4-kb PAM-addressable DNAtargets were prepared by preparative PGR amplification of plasmid-bomehuman sequences (here, a sequence from the human clathrin light chainCLTA gene). In a 20-uL reaction volume, 50 fmoles of linearized DNAtarget in the presence of 50 nM sgRNA, 39 nM recombinant purified Cas9protein (S. pyogenes; Agilent) and 10 mM MgCl₂ at pH 7.6 was incubatedat 37° C. for 30 min. Upon completion, 0.5 uL of RNace It (Agilent) wasadded, and incubation was continued at 37° C. for 5 min and then at 70°C. for 15 min. Subsequently 0.5 μL of Proteinase K (Mol. Bio. grade,NEB) was added and incubated at 37° C. for 15 min. Aliquots were loadedinto a DNA 7500 LabChip and were analyzed on a Bioanalyzer 2200. Theworkup steps served to release Cas9 from binding to target DNA, whichwere assayed for cleavage.

A series of guide RNAs as listed in FIG. 4 were chemically synthesized.Briefly, individual RNA strands were synthesized and HPLC purified. Alloligonucleotides were quality control approved on the basis of chemicalpurity by HPLC analysis and full-length strand purity by massspectrometry analysis. Each of these guide RNAs was designed to targetthe human CLTA gene.

The results are shown in FIG. 4. As shown in the Table 1 of FIG. 4, allbut one of the chemically synthesized guide RNAs targeted and cleavedthe CLTA-en coded DNA target sequence with significant cleavage rates.The one exception was “CLTA_37_Deoxy” guide RNA, which had a contiguoussequence of 37 deoxyribonucleotides at its 5′ end.

As disclosed herein, a variety of chemical modifications were tested atspecific positions in the sequence of a guide RNA. Surprisingly, thetested positions in the guide sequence of the guide RNA (a.k.a. thespacer sequence in the guide RNA) tolerated most of the modificationstested, including combinations of multiple modifications within singlenucleotides in the guide RNA, even when modifications were instantiatedin the target-binding sequences.

The results revealed that guide RNAs containing modifications atspecific positions were tolerated by active Cas protein and gRNA: Casprotein complexes, as the modifications did not prevent target-specificcleavage of the target polynucleotide. In all the guide RNA sequenceslisted in the Table 1 of FIG. 4, the first 20 nucleotides at the 5′ endare complementary to the target sequence in target DNA. Themodifications that were tested and found to be tolerated at specificpositions include 2′-O-methylribonucleotide (=2′OMe),2′-deoxyribonucleotide, racemic phosphorothioate internucleotidelinkage(s) (=P(S)), 3′-phosphonoacetate (=PACE), 3′-thiophosphonoacetate(=thioPACE), Z nucleotides, and combinations of these.

It is contemplated that the chemical modifications disclosed and testedherein, particularly at the tested positions (as listed in the Table 1of FIG. 4), will be tolerated at equivalent positions in a variety ofguide RNAs. In certain embodiments, the chemical modifications disclosedand tested herein are tolerated in any position in a guide RNA.

As disclosed herein; chemically modified nucleotides were incorporatedinto guide RNAs in an effort to improve certain properties. Suchproperties include improved nuclease resistance of the guide RNA,reduced off-target effects of a gRNA:Cas protein complex (also known asimproved specificity), improved efficacy of gRNA:Cas protein complexwhen cleaving, nicking or binding a target polynucleotide, improvedtransfection efficiency, and/or improved organelle localization such asnuclear localization.

While the use of modified RNA is known (e.g., to block nucleotlyticdegradation in certain applications), it is widely known that one cannotsimply incorporate modifications at any or all positions in an RNAsequence and expect it to function, particularly when the RNA sequenceneeds to complex with a protein or an enzyme to exert certain functions.Thus, it was not predictable whether these guide RNAs could toleratechemical modifications at a variety of nucleotide positions whileperforming sufficient or improved function in a CRISPR-Cas system. Infact, it was unexpected that the guide RNA can tolerate specificmodifications to the extent instantiated and tested, especially atseveral of the positions tested.

Example 2

To evaluate the ability of the chemically synthesized guide RNAs totarget and cleave a DNA target sequence, an in vitro cleavage assaysimilar to that described in Example 1 was used; Target DNA constructswere for human DNA targets (sequences from the human clathrin lightchain (CLTA1) gene, the human Interleukin 2 Receptor Gamma (IL2RG) gene,the human cytotoxic T-lymphocyte-associated protein 4 (CLTA4) gene, thehuman protocadherin alpha 4 (PCDHA4) gene, and the human engrailedhomeobox 1 (EN1) gene), along with off-target DNA constructs differingfrom the target DNA by one or more nucleotides.

Table 3 sets forth the guide RNA constructs and their sequences, alongwith DNA constructs used for assessing the ability of those guide RNAconstructs to target and cleave. In all the guide RNA sequences listedin the Table 3, the first 20 nucleotides at the 5′ end are complementaryto the target sequence in target DNA. ON target constructs comprise the20 nt target sequence. OFF target constructs comprise most of the same20 nucleotides as the target DNA, with 1, 2 or 3 nucleotide differences.Accordingly, the guide RNA is mostly, but not completely, complementaryto the sequence of the OFF target constructs. The OFF target constructsarc based on gene sequences known to occur in the human genome.

TABLE 3 Entry Target DNA RNA # Guide RNA Construct ConstructRNA sequence (5′→3′) length 2-piece dual-guide scaffoldUnmodified dual-guide RNA (dgRNA)   1 CLTA1 crRNA + tracrRNA CLTA1 ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGUCC 56 + targetCAAAAC (SEQ ID NO: 25) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)   2 CLTA1 crRNA +tracrRNA CLTA1 ON1- AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGUCC56 + target CAAAAC (SEQ ID NO: 25) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)   3 CLTA1 crRNA +tracrRNA CLTA1 OFF1- AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGUCC56 + target CAAAAC (SEQ ID NO: 25) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)   4 CLTA1 crRNA +tracrRNA CLTA1 OFF1- AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGUCC56 + target CAAAAC + (SEQ ID NO: 25)   86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)   5 CLTA1 crRNA +tracrRNA CLTA1 OFF2- AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGUCC56 + target CAAAAC (SEQ ID NO: 25) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)   6 CLTA1 crRNA +tracrRNA CLTA1 OFF2- AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGUCC56 + target CAAAAC (SEQ ID NO: 25) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)   7 CLTA1 crRNA +tracrRNA CLTA1 OFF3- AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGUCC56 + target CAAAAC (SEQ ID NO: 25) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)   8 CLTA1 crRNA +tracrRNA CLTA1 OFF3- AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGUCC56 + target CAAAAC (SEQ ID NO: 25) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)   9 CLTA1 crRNA +tracrRNA CLTA1 target AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGUCC56 + CAAAAC (SEQ ID NO: 25) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)  10 IL2RG_crRNA +tracrRNA IL2RGmg ON- UGGUAAUGAUGGCUUCAACAGUUUUAGAGCUAUGCUGUUUUGAAUGGUC56 + target CCAAAAC (SEQ ID NO: 27) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 28)Fluorophore-coupled dgRNA  11 CLTA1 crRNA + CLTA1 ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGUCC 56 +tracrRNA_aminoallyl- target CAAAAC (SEQ ID NO: 29) +  86 U57 + Cy5GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCA ACUUG(aminoallylU +Cy5)AAAAGUGGCACCGAGUCGGUGCUUUUU UU (SEQ ID NO: 30)2′OMethyl-modified dgRNA  12 IL2RG_crRNA_5′,3′- IL2RGmg ON- UGGUAAUGAUGGCUUCAACAGUUUUAGAGCUAUGCUGUUUUGAAUGGUC 56 + 3x(2′OMe) + targetCCA AAA C (SEQ ID NO: 31) +  86 tracrRNA_5′,3′- GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCA 3x(2′OMe)ACUUGUAAAAGUGGCACCGAGUCGGUGCUUU UUU U (SEQ ID NO: 32)2′OMethyl,3′Phosphorothioate-modified dgRNA  13 IL2RG_crRNA_5′,3′-IL2RGmg ON- U s G s G sUAAUGAUGGCUUCAACAGUUUUAGAGCUAUGCUGUUUUGAAUGG 56 +3x(2′OMe,3′P(S)) + target UCCCA A s A s A sC (SEQ ID NO: 33) +  86tracrRNA_5′,3′- G s G s AsACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAU  3x(2′OMe,3′P(S))CAACUUGUAAAAGUGGCACCGAGUCGGUGCUUU U s U s U sU  (SEQ ID NO: 34)2′OMethyl,3′PhosphorothioPACE-modified dgRNA  14 IL2RG_crRNA_5′,3′-IL2RGmg ON- U *s G *s G *sUAAUGAUGGCUUCAACAGUUUUAGAGCUAUGCUGUUUUGAAU56 + 3x(2′OMe, 3′thioPACE) + target GGUCCCA A *s A *s A*sC (SEQ ID NO: 35) +  86 tracrRNA_5′,3′- G *s G *s A*sACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGU 3x(2′OMe, 3′thioPACE)UAUCAACUUGUAAAAGUGGCACCGAGUCGGUGCUUU U *s U *s U *sU (SEQ ID NO: 36)  15IL2RG_crRNA_5′,3′- IL2RGmg ON- U*sGGUAAUGAUGGCUUCAACAGUUUUAGAGCUAUGCUGUUUUGAAUGGU 56 +1x(2′OMe, 3′thioPACE) + target CCCAAA A *sC (SEQ ID NO: 37) +  86tracrRNA_5′,3′- G *sGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAU 1x(2′OMe, 3′thioPACE) CAACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUU U *sU(SEQ ID NO: 38) 2-thioU-modified dgRNA  16 CLTA1_2thioU + CLTA1 ON1-AG(2sU)CCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGU 56 + 3 crRNA +tracrRNA target CCCAAAAC (SEQ ID NO: 39) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)  17 CLTA1_2thioU +CLTA1 ON1- AG(2sU)CCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGU 56 +3 crRNA + tracrRNA target CCCAAAAC (SEQ ID NO: 39) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)  18 CLTA1_2thioU +CLTA1 OFF1- AG(2sU)CCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGU  56 +3 crRNA + tracrRNA target CCCAAAAC (SEQ ID NO: 39) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)  19 CLTA1_2thioU +CLTA1 OFF1- AG(2sU)CCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGU 56 +3 crRNA + tracrRNA target CCCAAAAC (SEQ ID NO: 39) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)  20 CLTA1_2thioU +CLTA1 OFF2- AG(2sU)CCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGU 56 +3 crRNA + tracrRNA target CCCAAAAC (SEQ ID NO: 39) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)  21 CLTA1_2thioU +CLTA1 OFF2- AG(2sU)CCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGU  56 +3 crRNA + tracrRNA target CCCAAAAC (SEQ ID NO: 39) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)  22 CLTA1_2thioU +CLTA1 OFF3- AG(2sU)CCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGU 56 +3 crRNA + tracrRNA target CCCAAAAC (SEQ ID NO: 39) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)  23 CLTA1_2thioU +CLTA1 OFF3- AG(2sU)CCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGU 56 +3 crRNA + tracrRNA target CCCAAAAC (SEQ ID NO: 39) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)  24 CLTA1_2thioU +CLTA1 ON1- AGUCCUCA(2sU)CUCCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGU  56 +9 crRNA + tracrRNA target CCCAAAAC (SEQ ID NO: 40) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCA ACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)  25 CLTA1_2thioU +CLTA1 ON1- AGUCCUCA(2sU)CUCCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGU  56 +9 crRNA + tracrRNA target CCCAAAAC (SEQ ID NO: 40) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCA ACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)  26 CLTA1_2thioU +CLTA1 OFF1- AGUCCUCA(2sU)CUCCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGU  56 +9 crRNA + tracrRNA target CCCAAAAC (SEQ ID NO: 40) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCA ACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)  27 CLTA1_2thioU +CLTA1 OFF1- AGUCCUCA(2sU)CUCCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGU  56 +9 crRNA + tracrRNA target CCCAAAAC (SEQ ID NO: 40) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCA ACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)  28 CLTA1_2thioU +CLTA1 OFF2- AGUCCUCA(2sU)CUCCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGU  56 +9 crRNA + tracrRNA target CCCAAAAC (SEQ ID NO: 40) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCA ACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)  29 CLTA1_2thioU +CLTA1OFF2- AGUCCUCA(2sU)CUCCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGU  56 +9 crRNA + tracrRNA target CCCAAAAC (SEQ ID NO: 40) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCA ACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)  30 CLTA1_2thioU +CLTA1 OFF3- AGUCCUCA(2sU)CUCCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGU  56 +9 crRNA + tracrRNA target CCCAAAAC (SEQ ID NO: 40) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCA ACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)  31 CLTA1_2thioU +CLTA1 OFF3- AGUCCUCA(2sU)CUCCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGU  56 +9 crRNA + tracrRNA target CCCAAAAC (SEQ ID NO: 40) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCA ACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)  32 CLTA1_2thioU +CLTA1 ON1- AGUCCUCAUC(2sU)CCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGU  56 +11 crRNA + tracrRNA target CCCAAAAC (SEQ ID NO: 41) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCA ACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)  33 CLTA1_2thioU +CLTA1 ON1- AGUCCUCAUC(2sU)CCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGU  56 +11 crRNA + tracrRNA target CCCAAAAC (SEQ ID NO: 41) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCA ACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)  34 CLTA1_2thioU +CLTA1 OFF1- AGUCCUCAUC(2sU)CCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGU  56 +11 crRNA + tracrRNA target CCCAAAAC (SEQ ID NO: 41) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCA ACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)  35 CLTA1_2thioU +CLTA1 OFF1- AGUCCUCAUC(2sU)CCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGU  56 +11 crRNA + tracrRNA target CCCAAAAC (SEQ ID NO: 41) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCA ACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)  36 CLTA1_2thioU +CLTA1 OFF2- AGUCCUCAUC(2sU)CCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGU  56 +11 crRNA + tracrRNA target CCCAAAAC (SEQ ID NO: 41) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCA ACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)  37 CLTA1_2thioU + ″AGUCCUCAUC(2sU)CCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGU  56 + 11 crRNA +tracrRNA CCCAAAAC (SEQ ID NO: 41) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCA ACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)  38 CLTA1_2thioU +CLTA1 OFF3- AGUCCUCAUC(2sU)CCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGU  56 +11 crRNA + tracrRNA target CCCAAAAC (SEQ ID NO: 41) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCA ACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)  39 CLTA1_2thioU + ″AGUCCUCAUC(2sU)CCCUCAAGCGUUUAAGAGCUAUGCUGUUUUGAAUGGU  56 + 11 crRNA +tracrRNA CCCAAAAC (SEQ ID NO: 41) +  86GGAACCAUUCAAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCA ACUUGUAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 26)Single-guide scaffold Unmodified single-guide RNA (sgRNA)  40CLTA1 sgRNA (Batch #1) CLTA1 ON1-AGUCCUCAUCUCCCUCAA6CGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 42)  41 CLTA1 sgRNA (Batch #1) CLTA1 ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 42)  42 CLTA1 sgRNA (Batch #2) CLTA1 ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 42)  43 CLTA1 sgRNA (Batch #2) CLTA1 ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 42)  44 CLTA1 sgRNA (Batch #3) CLTA1 ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 42)  45 CLTA1 sgRNA (Batch #3) CLTA1 ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 42)  46 CLTA1 SgRNA (Batch #3) CLTA1 ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 42)  47 CLTA1 sgRNA (Batch #3) CLTA1mg ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 42)  48 CLTA1 sgRNA (Batch #3) CLTA1mg ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 42)  49 CLTA1 sgRNA (Batch #3) CLTA1mg ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 42)  50 CLTA1 sgRNA (Batch #3) CLTA1mg OFF1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 42)  51 CLTA1 sgRNA (Batch #3) CLTA1mg OFF3-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 42)  52 CLTA1 sgRNA (crude) CLTA1 ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 42)  53 CLTA1_Bos sgRNA CLTA1mg ON1-AGUCCUCAUCUCCCUCAAGCGUUUUAGAGCUAGUAAUAGCAAGUUAAAAUA  100 targetAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU  (SEQ ID NO: 13)  54CLTA1_Bos sgRNA CLTA1mg ON1-AGUCCUCAUCUCCCUCAAGCGUUUUAGAGCUAGUAAUAGCAAGUUAAAAUA  100 targetAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU  (SEQ ID NO: 43)  55CLTA1_Bos sgRNA CLTA1mg ON1-AGUCCUCAUCUCCCUCAAGCGUUUUAGAGCUAGUAAUAGCAAGUUAAAAUA  100 targetAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU  (SEQ ID NO: 43)  56CLTA1_Bos sgRNA CLTA1mg OFF1-AGUCCUCAUCUCCCUCAAGCGUUUUAGAGCUAGUAAUAGCAAGUUAAAAUA  100 targetAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU  (SEQ ID NO: 43)  57CLTA1_Bos sgRNA CLTA1mg OFF3-AGUCCUCAUCUCCCUCAAGCGUUUUAGAGCUAGUAAUAGCAAGUUAAAAUA  100 targetAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU  (SEQ ID NO: 43)  58CLTA4 SgRNA CLTA4 ON-GCAGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAUAGC  113 targetAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 44)  59 CLTA4 sgRNA CLTA4 ON-GCAGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAUAGC  113 targetAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 44)  60 CLTA4 sgRNA CLTA4 ON-GCAGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAUAGC  113 targetAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 44)  61 CLTA4 sgRNA CLTA4mg ON-GCAGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAUAGC  113 targetAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 44)  62 CLTA4 sgRNA CLTA4mg ON-GCAGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAUAGC  113 targetAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 44)  63 CLTA4 sgRNA CLTA4mg OFF5-GCAGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAUAGC  113 targetAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 44)  64 CLTA1_Truncated_18mer CLTA1mg ON1-UCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCAAG  111 targetUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGG UGCUUUUUUU (SEQ ID NO: 45)  65 CLTA1_Truncated_18mer CLTA1mg ON1-UCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCAAG  111 targetUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGG UGCUUUUUUU (SEQ ID NO: 45)  66 CLTA1_Truncated_18mer CLTA1mg OFF1-UCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCAAG  111 targetUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGG UGCUUUUUUU (SEQ ID NO: 45)  67 CLTA1_Truncated_18mer CLTA1mg OFF3-UCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCAAG  111 targetUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGG UGCUUUUUUU (SEQ ID NO: 45)  68 CLTA1_Truncated_17mer CLTA1mg ON1-CCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCAAGU  110 targetUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCUUUUUUU (SEQ ID NO: 46)  69 CLTA1_Truncated_17mer CLTA1mg ON1-CCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCAAGU  110 targetUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCUUUUUUU (SEQ ID NO: 46)  70 CLTA1_Truncated_17mer CLTA1mg OFF1-CCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCAAGU  110 targetUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCUUUUUUU (SEQ ID NO: 46)  71 CLTA1_Truncated_17mer CLTA1mg OFF3-CCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCAAGU  110 targetUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCUUUUUUU (SEQ ID NO: 46)  72 CLTA1_1xExtraG CLTA1mg ON1-GAGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGC  114 targetAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 47)  73 CLTA1_1xExtraG CLTA1mg ON1-GAGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGC  114 targetAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 47)  74 CLTA1_1xExtraG CLTA1mg OFF1-GAGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGC  114 targetAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 47)  75 CLTA1_1xExtraG CLTA1mg OFF3-GAGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGC  114 targetAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 47)  76 CLTA1_2xExtraG CLTA1mg ON1-GGAGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAG  115 targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUUUUU (SEQ ID NO: 48)  77 CLTA1_2xExtraG CLTA1mg ON1-GGAGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAG  115 targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUUUUU (SEQ ID NO: 48)  78 CLTA1_2xExtraG CLTA1mg OFF1-GGAGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAG  115 targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUUUUU (SEQ ID NO: 48)  79 CLTA1_2xExtraG CLTA1mg OFF3-GGAGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAG  115 targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUUUUU (SEQ ID NO: 48)  80 CLTA1_63U,64U CLTA1mg ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAUUCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 49)  81 CLTA1_63A,64A CLTA1mg ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAAACUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 50)  82 CLTA1_63A,64A, CLTA1mg ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 70U,71U targetAGUUUAAAUAAAACUAGUUUGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 51)  83 CLTA1_cis-block(1- CLTA1mg ON1-GGACUUUUUUUAGUCCUCAUCUCCCUCAAGCGUUUUAGAGCUAGAAAUAG  111 5)_polyU_sgRNAtarget CAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUU (SEQ ID NO: 52)  84 CLTA1_cis-block(1- CLTA1mg ON1-GGACUUUUUUUAGUCCUCAUCUCCCUCAAGCGUUUUAGAGCUAGAAAUAG  111 5)_polyU_sgRNAtarget CAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUU (SEQ ID NO: 52)  85 CLTA1_cis-block(1- CLTA1mg OFF1-GGACUUUUUUUAGUCCUCAUCUCCCUCAAGCGUUUUAGAGCUAGAAAUAG  111 5)_polyU_sgRNAtarget CAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUU (SEQ ID NO: 52)  86 CLTA1_cis-block(1- CLTA1mg OFF3-GGACUUUUUUUAGUCCUCAUCUCCCUCAAGCGUUUUAGAGCUAGAAAUAG  111 5)_polyU_sgRNAtarget CAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUU (SEQ ID NO: 52)  87 CLTA1_cis-block(1- CLTA1mg ON1-GAUGAGGACUUUUUUUAGUCCUCAUCUCCCUCAAGCGUUUUAGAGCUAGA  116 10)_polyU_sgRNAtarget AAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUUUU (SEQ ID NO: 53)  88 CLTA1_cis-block(1- CLTA1mg ON1-GAUGAGGACUUUUUUUAGUCCUCAUCUCCCUCAAGCGUUUUAGAGCUAGA  116 10)_polyU_sgRNAtarget AAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUUUU (SEQ ID NO: 53)  89 CLTA1_cis-block(1- CLTA1mg OFF1-GAUGAGGACUUUUUUUAGUCCUCAUCUCCCUCAAGCGUUUUAGAGCUAGA  116 10)_polyU_sgRNAtarget AAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUUUU (SEQ ID NO: 53)  90 CLTA1_cis-block(1- CLTA1mg OFF3-GAUGAGGACUUUUUUUAGUCCUCAUCUCCCUCAAGCGUUUUAGAGCUAGA  116 10)_polyU_sgRNAtarget AAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUUUU (SEQ ID NO: 53)  91 CLTA1_cis-block(16- CLTA1mg ON1-GCUUGUUUUUUAGUCCUCAUCUCCCUCAAGCGUUUUAGAGCUAGAAAUAG  111 20)_polyU_sgRNAtarget CAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUU (SEQ ID NO: 54)  92 CLTA1_cis-block(16- CLTA1mg ON1-GCUUGUUUUUUAGUCCUCAUCUCCCUCAAGCGUUUUAGAGCUAGAAAUAG  111 20)_polyU_sgRNAtarget CAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUU (SEQ ID NO: 54)  93 CLTA1_cis-block(16- CLTA1mg OFF1-GCUUGUUUUUUAGUCCUCAUCUCCCUCAAGCGUUUUAGAGCUAGAAAUAG  111 20)_polyU_sgRNAtarget CAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUU (SEQ ID NO: 54)  94 CLTA1_cis-block(16- CLTA1mg OFF3-GCUUGUUUUUUAGUCCUCAUCUCCCUCAAGCGUUUUAGAGCUAGAAAUAG  111 20)_polyU_sgRNAtarget CAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUU (SEQ ID NO: 54) DMT-modified sgRNA  95 CLTA1_DMT-ON SgRNACLTA1 ON1- (dmt)AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAU  113target AGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG AGUCGGUGCUUUUUUU (SEQ ID NO: 55)  96 CLTA1_DMT-ON/ CLTA1 ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 OFF sgRNAtarget AGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 56) Fluorophore-modified sgRNA  97CLTA1_IntFl_sgLoop CLTA1 ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGG(Fl)AACAGCAUAG  113 targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 57)  98 CLTA1_IntFl_sgLoop CLTA1mg ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGG(Fl)AACAGCAUAG  113 targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 57)  99 CLTA1_IntFl_sgLoop CLTA1mg ON1-AGUCCUCAUCUCCCUCAA6CGUUUAAGAGCUAUGCUGG(Fl)AACAGCAUAG  113 targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 57) 100 CLTA1_IntFl_sgLoop CLTA1mg OFF1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGG(Fl)AACAGCAUAG  113 targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 57) 101 CLTA1_IntFl_sgLoop CLTA1mg OFF3-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGG(Fl)AACAGCAUAG  113 targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 57) 102 CLTA1_IntFl_sgLoop_5′,3′- CLTA1mg ON1-AGU CCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGG(Fl)AACAGCAUAG  113 3x(2′OMe)target CAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU  CGGUGCUUUUUU U (SEQ ID NO: 58) 103 CLTA1_IntFl_sgLoop_5′,3′- CLTA1mg ON1- AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGG(Fl)AACAGCAUAG  113 3x(2′OMe) targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU  CGGUGCUUU UUUU (SEQ ID NO: 58) 104 CLTA1_IntFl_sgLoop_5′,3′- CLTA1mg OFF1- AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGG(Fl)AACAGCAUAG  113 3x(2′OMe) targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU  CGGUGCUUU UUUU (SEQ ID NO: 58) 105 CLTA1_IntFl_sgLoop_5′,3′- CLTA1mg OFF3- AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGG(Fl)AACAGCAUAG  113 3x(2′OMe) targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU  CGGUGCUUU UUUU (SEQ ID NO: 58) 106 CLTA1_IntFl_sgLoop_5′,3′- CLTA1mg ON1- A s G s UsCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGG(Fl)AACAGCA  113 3x(2′OMe, 3′P(S))target UAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGA  GUCGGUGCUUUU s U s U sU (SEQ ID NO: 59) 107 CLTA1_IntFl_sgLoop_5′,3′- CLTA1mg ON1-A s G s U sCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGG(Fl)AACAGCA  1133x(2′OMe, 3′P(S)) targetUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGA  GUCGGUGCUUU U s Us U sU (SEQ ID NO: 59) 108 CLTA1_IntFl_sgLoop_5′,3′- CLTA1mg OFF1- A s Gs U sCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGG(Fl)AACAGCA  1133x(2′OMe, 3′P(S)) targetUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGA  GUCGGUGCUUU U s Us U sU (SEQ ID NO: 59) 109 CLTA1_IntFl_sgLoop_5′,3′- CLTA1mg OFF3- A s Gs U sCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGG(Fl)AACAGCA  1133x(2′OMe, 3′P(S)) targetUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGA  GUCGGUGCUUU U s Us U sU (SEQ ID NO: 59) 110 CLTA1_IntFl_sgLoop_5′,3′- CLTA1mg ON1- A *s G*s U *sCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGG(Fl)AACA  1133x(2′OMe, 3′thioPACE) targetGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCAC  CGAGUCGGUGCUUU U*s U *s U *sU (SEQ ID NO: 60) 111 CLTA1_IntFl_sgLoop_5′,3′- CLTA1mg ON1-A *s G *s U *sCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGG(Fl)AACA  1133x(2′OMe, 3′thioPACE) targetGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCAC  CGAGUCGGUGCUUU U*s U *s U *sU (SEQ ID NO: 60) 112 CLTA1_IntFl_sgLoop_5′,3′-CLTA1mg OFF1- A *s G *s U *sCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGG(Fl)AACA 113 3x(2′OMe, 3′thioPACE) targetGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCAC  CGAGUCGGUGCUUU U*s U *s U *sU (SEQ ID NO: 60) 113 CLTA1_IntFl_sgLoop_5′,3′-CLTA1mg OFF3- A *s G *s U *sCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGG(Fl)AACA 113 3x(2′OMe, 3′thioPACE) targetGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCAC  CGAGUCGGUGCUUU U*s U *s U *sU (SEQ ID NO: 60) 114 CLTA4_3xFl- CLTA4mg ON- U _(o) *s(Fl_(o) )GCAGAUGUAGUGUUUCCACAGUUUaAGAGCUAGUAAUAGCAAGU  102 Int_3x(2′OMe,target UuAAAUAAGGCUAGUCCGUUA(Fl)CAACUUGAAAAAGUGGCACCGAGUCGG  3′thioPACE)UGCU(Fl) U *sU (SEQ ID NO: 61) 115 CLTA4_3xFl- CLTA4mg OFF5- U _(o)*s(Fl _(o) )GCAGAUGUAGUGUUUCCACAGUUUaAGAGCUAGUAAUAGCAAGU  102Int_3x(2′OMe, targetUuAAAUAAGGCUAGUCCGUUA(Fl)CAACUUGAAAAAGUGGCACCGAGUCGG  3′thioPACE)UGCU(Fl) U *sU (SEQ ID NO: 61) 116 CLTA4_3xFl- CLTA4mg ON- G*sCAGAUGUAGUGUUUCCACAGUUUaAGAGCUAG(Fl)AAUAGCAAGUUu  100 Loops_3x(2′OMe,target AAAUAAGGCUAGUCCGUUAUCAACUUG(Fl)AAAAGUGGCACCGAG(Fl)C  3′thioPACE)GGUGCUU U *sU (SEQ ID NO: 62) 117 CLTA4_3xFl- CLTA4mg OFF5- G*sCAGAUGUAGUGUUUCCACAGUUUaAGAGCUAG(Fl)AAUAGCAAGUUu  100 Loops_3x(2′OMe,target AAAUAAGGCUAGUCCGUUAUCAACUUG(Fl)AAAAGUGGCACCGAG(Fl)C  3′thioPACE)GGUGCUU U *sU (SEQ ID NO: 62) 3′Phosphorothioate-modified sgRNA 118CLTA1_5′-2xP(S) sgRNA CLTA1 ON1-AsGsUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGAAACAGCAUAGC  113 targetAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 63) 119 CLTA1_5′-3xP(S) sgRNA CLTA1 ON1-AsGsUsCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGAAACAGCAUAG  113 targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUUUUU (SEQ ID NO: 64) 120 CLTA1_5′-4xP(S) sgRNA CLTA1 ON1-AsGsUsCsCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGAAACAGCAUA  113 targetGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGA GUCGGUGCUUUUUUU (SEQ ID NO: 65) 121 CLTA1_3′-4xP(S) SgRNA CLTA1 ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGAAACAGCAUAGCA  113 targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUsUsUsUsU (SEQ ID NO: 66) 2′OMethyl-modified sgRNA 122CLTA1_2′OMe + CLTA1 ON1- AGUCCUCAUCUCCCUCAAG CGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 20 sgRNA targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 67) 123 CLTA1_2′OMe + CLTA1 ON1-AGUCCUCAUCUCCCUCAA G CGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 19 sgRNAtarget AGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 68) 124 CLTA1_2′OMe + CLTA1mg ON1-AGUCCUCAUCUCCCUCAA G CGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 19 sgRNAtarget AGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 68) 125 CLTA1_2′OMe + CLTA1mg ON1-AGUCCUCAUCUCCCUCAA G CGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 19 sgRNAtarget AGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 68) 126 CLTA1_2′OMe + CLTA1mg OFF1-AGUCCUCAUCUCCCUCAA G CGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 19 sgRNAtarget AGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 68) 127 CLTA1_2′OMe + CLTA1mg OFF3-AGUCCUCAUCUCCCUCAA G CGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 19 sgRNAtarget AGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 68) 128 CLTA1_2′OMe + CLTA1 ON1-AGUCCUCAUCUCCCUCA A GCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 18 sgRNAtarget AGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 69) 129 CLTA1_2′OMe + CLTA1mg ON1-AGUCCUCAUCUCCCUCA A GCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 18 sgRNAtarget AGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 69) 130 CLTA1_2′OMe + CLTA1mg ON1-AGUCCUCAUCUCCCUCA A GCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 18 sgRNAtarget AGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 69) 131 CLTA1_2′OMe + CLTA1mg OFF1-AGUCCUCAUCUCCCUCA A GCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 18 sgRNAtarget AGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 69) 132 CLTA1_2′OMe + CLTA1mg OFF3-AGUCCUCAUCUCCCUCA A GCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 18 sgRNAtarget AGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 69) 133 CLTA1_2′OMe + CLTA1 ON1-AGUCCUCAUCUCCCUCA A GCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 17 sgRNAtarget AGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 70) 134 CLTA1_2′OMe + CLTA1mg ON1-AGUCCUCAUCUCCCUC A AGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 17 sgRNAtarget AGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 70) 135 CLTA1_2′OMe + CLTA1mg ON1-AGUCCUCAUCUCCCUC A AGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 17 sgRNAtarget AGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 70) 136 CLTA1_2′OMe + CLTA1mg OFF1-AGUCCUCAUCUCCCUC A AGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 17 sgRNAtarget AGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 70) 137 CLTA1_2′OMe + CLTA1mg OFF3-AGUCCUCAUCUCCCUC A AGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 17 sgRNAtarget AGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 70) 138 CLTA1_2′OMe + CLTA1 ON1-AGUCCUCAUCUCCCUC AA GCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 17,18 sgRNAtarget AGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 71) 139 CLTA1_2′OMe + CLTA1mg ON1-AGUCCUCAUCUCCCUC AA GCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 17,18 sgRNAtarget AGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 71) 140 CLTA1_2′OMe + CLTA1mg ON1-AGUCCUCAUCUCCCUC AA GCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 17,18 sgRNAtarget AGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 71) 141 CLTA1_2′OMe + CLTA1mg OFF1-AGUCCUCAUCUCCCUC AA GCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 17,18 sgRNAtarget AGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 71) 142 CLTA1_2′OMe + CLTA1mg OFF3-AGUCCUCAUCUCCCUC AA GCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 17,18 sgRNAtarget AGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 71) 143 CLTA1_5′,3′- CLTA1 ON1- AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 3x(2′OMe) sgRNAtarget AGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC  GGUGCUUU UUUU (SEQ ID NO: 72) 144 CLTA4_5′,3′- CLTA4mg ON- GCAGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAUAGC  113 3x(2′OMe) sgRNAtarget AAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU  CGGUGCUUU UUUU (SEQ ID NO: 73) 145 CLTA4_5′,3′- CLTA4mg OFF5- GCAGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAUAGC  113 3x(2′OMe) sgRNAtarget AAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU  CGGUGCUUU UUUU (SEQ ID NO: 73) 146 CLTA1_5′- CLTA1 ON1- AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 20x(2′OMe) sgRNA targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 74) 147 CLTA1_5′- CLTA1mg ON1-AGUCCUCAUCUCCCUCAAGC GUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  11320x(2′OMe) sgRNA targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 74) 148 CLTA1_5′- CLTA1mg ON1-AGUCCUCAUCUCCCUCAAGC GUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  11320x(2′OMe) sgRNA targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 74) 149 CLTA1_5′- CLTA1mg OFF1-AGUCCUCAUCUCCCUCAAGC GUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  11320x(2′OMe) sgRNA targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 74) 150 CLTA1_5′- CLTA1mg OFF3-AGUCCUCAUCUCCCUCAAGC GUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  11320x(2′OMe) sgRNA targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 74) 151 CLTA1_5′- CLTA1 ON1-AGUCCUCAUCUCCCUCAAGCGUUUAA GAGCUAUGCUGGUAACAGCAUAGC  11326x(2′OMe) sgRNA targetAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 75) 152 CLTA1_5′- CLTA1 ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUG GUAACAGCAUAGC  11337x(2′OMe) SgRNA targetAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 76) 153 CLTA1_41x(2′OMeC/U)_QB3 CLTA1mg ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 77) 154 CLTA1_47x(2′OMeC/U)_QB3 CLTA1 ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 78) 155 CLTA1_47x(2′OMeC/U)_QB3 CLTA1mg ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 79) 156 CLTA1_47x(2′OMeC/U)_QB3 CLTA1mg ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 79) 157 CLTA1_47x(2′OMeC/U)_QB3 CLTA1mg OFF1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 79) 158 CLTA1_47x(2′OMeC/U)_QB3 CLTA1mg OFF3-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 79) 159 CLTA1_47x(2′OMeG/A)_QB3 CLTA1 ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 80) 160 CLTA1_47x(2′OMeG/A)_QB3 CLTA1mg ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGA)AAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 80) 161 CLTA1_47x(2′OMeG/A)_QB3 CLTA1mg ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 80) 162 CLTA1_47x(2′OMeG/A)_QB3 CLTA1mg OFF1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 80) 163 CLTA1_47x(2′OMeG/A)_QB3 CLTA1mg OFF3-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 80) 164 CLTA1_43x(2′OMeG/A)_BOS CLTA1 ON1-AGUCCUCAUCUCCCUCAAGCGUUUUAGAGCUAGUAAUAGCAAGUUAAAAUA  100 targetAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU  (SEQ ID NO: 81) 165CLTA1_43x(2′OMeG/A)_Bos CLTA1mg ON1-AGUCCUCAUCUCCCUCAAGCGUUUUAGAGCUAGUAAUAGCAAGUUAAAAUA  100 targetAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU  (SEQ ID NO: 81) 166CLTA1_43x(2′OMeG/A)_Bos CLTA1mg ON1-AGUCCUCAUCUCCCUCAAGCGUUUUAGAGCUAGUAAUAGCAAGUUAAAAUA  100 targetAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU  (SEQ ID NO: 81) 167CLTA1_43x(2′OMeG/A)_Bos CLTA1mg OFF1-AGUCCUCAUCUCCCUCAAGCGUUUUAGAGCUAGUAAUAGCAAGUUAAAAUA  100 targetAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU  (SEQ ID NO: 81) 168CLTA1_43x(2′OMeG/A)_Bos CLTA1mg OFF3-AGUCCUCAUCUCCCUCAAGCGUUUUAGAGCUAGUAAUAGCAAGUUAAAAUA  100 targetAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU  (SEQ ID NO: 81) 169CLTA4 sgRNA_5′,3′- CLTA4 ON- GCAGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAUAGC  113 3x(2′OMe) targetAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU  CGGUGCUUU UUUU (SEQ ID NO: 82) 170 CLTA4 sgRNA_5′,3′- CLTA4 ON- GCAGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAUAGC  113 3x(2′OMe) targetAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU  CGGUGCUUU UUUU (SEQ ID NO: 82) 171 CLTA4_47x(2′OMeC/U)_QB3 CLTA4 ON-GCAGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGUAACAGCAUAGC  113 targetAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 83) 172 CLTA4_47x(2′OMeC/U)_QB3 CLTA4 ON-GCAGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGUAACAGCAUAGC  113 targetAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 83) 173 CLTA4_47x(2′OMeC/U)_QB3 CLTA4 ON-GCAGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGUAACAGCAUAGC  113 targetAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 83) 174 CLTA4_49x(2′OMeG/A)_Bos CLTA4 ON-GCAGAUGUAGUGUUUCCACAGUUUUAGAGCUAGUAAUAGCAAGUUAAAAU  100 targetAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU  (SEQ ID NO: 84) 175CLTA4_49x(2′OMeG/A)_Bos CLTA4 ON-GCAGAUGUAGUGUUUCCACAGUUUUAGAGCUAGUAAUAGCAAGUUAAAAU  100 targetAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU  (SEQ ID NO: 84) 176CLTA4_49x(2′OMeG/A)_Bos CLTA4 ON-GCAGAUGUAGUGUUUCCACAGUUUUAGAGCUAGUAAUAGCAAGUUAAAAU  100 targetAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU  (SEQ ID NO: 84) 177CLTA4_39x(2′OMeC/U)_BoS CLTA4 ON-GCAGAUGUAGUGUUUCCACAGUUUUAGAGCUAGUAAUAGCAAGUUAAAAU  100 targetAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU  (SEQ ID NO: 85) 178CLTA4_39x(2′OMeC/U)_BoS CLTA4 ON-GCAGAUGUAGUGUUUCCACAGUUUUAGAGCUAGUAAUAGCAAGUUAAAAU  100 targetAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU  (SEQ ID NO: 85) 179CLTA4_39x(2′OMeC/U)_BoS CLTA4 ON-GCAGAUGUAGUGUUUCCACAGUUUUAGAGCUAGUAAUAGCAAGUUAAAAU  100 targetAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU  (SEQ ID NO: 85)2′Deoxy-modified sgRNA 180 CLTA1_5′-20x(2′deoxy) CLTA1 ON1-AGTCCTCATCTCCCTCAAGC GUUUAAGAGCUAUGCUGGUAACAGCAUAG  113 targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUUUUU (SEQ ID NO: 86) 181 CLTA1_5′-26x(2′deoxy) CLTA1 ON1-AGTCCTCATCTCCCTCAAGCGTTTAA GAGCUAUGCUGGUAACAGCAUAG 113 targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 87) 182 CLTA1_5′-37x(2′deoxy) CLTA1 ON1-AGTCCTCATCTCCCTCAAGCGTTTAAGAGCTATGCTG GUAACAGCAUA  113 targetGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUUUUU (SEQ ID NO: 88) 2′Deoxy,3′PACE-modified sgRNA 183CLTA4_2′deoxy3′PACE + 15 CLTA4mg ON- GCAGAUGUAGUGUU U*CCACAGUUUAAGAGCUAUGCUGGUAACAGCAUAG  113 targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUUUUU (SEQ ID NO: 89) 184 CLTA4_2′deoxy3′PACE + 15CLTA4mg OFF5- GCAGAUGUAGUGUU U *CCACAGUUUAAGAGCUAUGCUGGUAACAGCAUAG  113target CAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUUUUU (SEQ ID NO: 89) 2′OMethyl,3′PACE-modified sgRNA 1855′-1x(2′OMe, CLTA1mg ON1-A*GUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGC  1133′PACE)_CLTA1 sgRNA targetAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 90) 186 5′-1x(2′OMe, CLTA1mg ON1-A*GUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGC  1133′PACE)_CLTA1 sgRNA targetAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 90) 187 5′-2x(2′OMe, CLTA1 ON1-A*G*UCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAG  1133′PACE)_CLTA1 sgRNA targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUUUUU (SEQ ID NO: 91) 188 5′-2x(2′OMe, CLTA1 ON1-A*G*UCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAG  1133′PACE)_CLTA1 sgRNA targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUUUUU (SEQ ID NO: 91) 189 5′-2x(2′OMe, CLTA1mg ON1-A*G*UCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAG  1133′PACE)_CLTA1 sgRNA targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUUUUU (SEQ ID NO: 91) 190 5′-2x(2′OMe, CLTA1mg ON1-A*G*UCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAG  1133′PACE)_CLTA1 sgRNA targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUUUUU (SEQ ID NO: 91) 191 5′-3x(2′OMe, CLTA1mg ON1-G*G*A*GUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCA  1153′PACE)_CLTA1 sgRNA targetUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC GAGUCGGUGCUUUUUUU (SEQ ID NO: 92) 192 5′-3x(2′OMe, CLTA1mg ON1-G*G*A*GUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCA  1153′PACE)_CLTA1 sgRNA targetUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC GAGUCGGUGCUUUUUUU (SEQ ID NO: 92) 193 5′-4x(2′OMe, CLTA1 ON1-A*G*U*C*CUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAU  1133′PACE)_CLTA1 sgRNA targetAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG AGUCGGUGCUUUUUUU (SEQ ID NO: 93) 194 5′-4x(2′OMe, CLTA1mg ON1-A*G*U*C*CUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAU  1133′PACE)_CLTA1 sgRNA targetAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG AGUCGGUGCUUUUUUU (SEQ ID NO: 93) 195 5′-4x(2′OMe, CLTA1mg ON1-A*G*U*C*CUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAU  1133′PACE)_CLTA1 sgRNA targetAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG AGUCGGUGCUUUUUUU (SEQ ID NO: 93) 196 5′-5x(2′OMe, CLTA1mg ON1-G*G*A*G*U*CCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAG  1153′PACE)_CLTA1 sgRNA targetCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUUUUUUU (SEQ ID NO: 94) 197 5′-5x(2′OMe, CLTA1mg ON1-G*G*A*G*U*CCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAG  1153′PACE)_CLTA1 sgRNA targetCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCUUUUUUU (SEQ ID NO: 94) 198 CLTA1_3′- CLTA1 ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  1134x(2′OMe,3′PACE) SgRNA targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUU*U*U*U*U (SEQ ID NO: 95) 199 CLTA1_3′- CLTA1 ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  1134x(2′OMe,3′PACE) SgRNA targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUU*U*U*U*U (SEQ ID NO: 95) 200 CLTA1_3′- CLTA1mg ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  1134x(2′OMe,3′PACE) sgRNA targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUU*U*U*U*U (SEQ ID NO: 95) 201 CLTA1_3′- CLTA1mg ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  1134x(2′OMe,3′PACE) sgRNA targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUU*U*U*U*U (SEQ ID NO: 95) 202 CLTA1_3′- CLTA1 ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  1135x(2′OMe,3′PACE) sgRNA targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUU*U*U*U*U*U (SEQ ID NO: 96) 203 CLTA1_3′- CLTA1 ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA 1135x(2′OMe,3′PACE) sgRNA targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUU*U*U*U*U*U (SEQ ID NO: 96) 204 CLTA1_3′- CLTA1mg ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  1135x(2′OMe,3′PACE) sgRNA targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUU*U*U*U*U*U(SEQ ID NO: 96) 205 CLTA1_3′- CLTA1mg ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  1135x(2′OMe,3′PACE) sgRNA targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUU*U*U*U*U*U (SEQ ID NO: 96) 206 5′-3x(2′OMe, CLTA1 ON1- C_(o)*A*G*UCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAU  1143′PACE)_plus1 target AGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG overhg CLTA1 AGUCGGUGCUUUUUUU (SEQ ID NO: 97) 207 5′-3x(2′OMe,CLTA1mg ON1- C _(o)*A*G*UCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAU 114 3′PACE)_plus1  targetAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG  overhg_CLTA1AGUCGGUGCUUUUUUU (SEQ ID NO: 97) 208 5′-3x(2′OMe, CLTA1mg ON1- C_(o)*A*G*UCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAU  1143′PACE)_plus1 target AGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG overhg_CLTA1 AGUCGGUGCUUUUUUU (SEQ ID NO: 97) 2095′-3x(2′OMe,3′PACE)_plus1 CLTA1 ON1- G_(o)*A*G*UCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACA6CAU  114NC overhg_CLTA1 targetAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG AGUCGGUGCUUUUUUU (SEQ ID NO: 98 210 5′-3x(2′OMe,3′PACE)_plus1 CLTA1mg ON1- G _(o)*A*G*UCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAU 114 NC_overhg_CLTA1 targetAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG AGUCGGUGCUUUUUUU (SEQ ID NO: (SEQ ID NO: 98) 2115′-3x(2′OMe,3′PACE)_plus1  CLTA1mg ON1- G_(o)*A*G*UCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAU  114NC_overhg_CLTA1 targetAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG AGUCGGUGCUUUUUUU (SEQ ID NO: 98) 212 5′-5x(2′OMe,3′PACE)_plus2CLTA1 ON1- U _(o)*C _(o)*A*G*U*CCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACA 115 overhg_CLTA1 targetGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 99) 213 5′-5x(2′OMe,3′PACE)_plus2CLTA1mg ON1- U _(o)*C_(o)*A*G*U*CCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACA  115 overhg_CLTA1target GCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 99) 214 5′-5x(2′OMe,3′PACE) plus2CLTA1mg ON1- U _(o)*C_(o)*A*G*U*CCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACA  115 overhg_CLTA1target GCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 99) 215 5′-5x(2′OMe,3′PACE)_plus2CLTA1 ON1- A _(o)*G _(o)*A*G*U*CCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACA 115 NC_overhg_CLTA1 targetGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 100) 216 5′-5x(2′OMe,3′PACE)_plus2CLTA1mg ON1- A _(o)*G_(o)*A*G*U*CCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACA  115 NC_overhg_CLTA1target GCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 100) 217 5′-5x(2′OMG,3′PACE)_plus2 CLTA1mg ON1- A _(o)*G_(o)*A*G*U*CCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACA  115 NC_overhg_CLTA1target GCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 100) 218 5′-7x(2′OMe,3′PACE)_plus3 CLTA1 ON1- C _(o)*U _(o)*C_(o)*A*G*U*C*CUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUA  116 overhg_CLTA1_3′-target ACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU 4x(2′OMe,3′PACE) GGCACCGAGUCGGUGCUUU*U*U*U*U (SEQ ID NO: 101) 2195′-7x(2′OMe,3′PACE)_plus3  CLTA1mg ON1- C _(o)*U _(o)*C_(o)*A*G*U*C*CUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUA  116 overhg_CLTA1_3′-target ACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU 4x(2′OMe,3′PACE) GGCACCGAGUCGGUGCUUU*U*U*U*U (SEQ ID NO: 101) 2205′-7x(2′OMe,3′PACE)_plus3  CLTA1mg ON1- C _(o)*U _(o)*C_(o)*A*G*U*C*CUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUA  116 overhg_CLTA1_3′-target ACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU 4x(2′OMe,3′PACE) GGCACCGAGUCGGUGCUUU*U*U*U*U (SEQ ID NO: 101) 2215′-7x(2′OMe,3′PACE)_plus3 CLTA1 ON1- G _(o)*A _(o)*G_(o)*A*G*U*C*CUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUA  116NC_overhg_CLTA1_3′- targetACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU  4x(2′OMe,3′PACE)GGCACCGAGUCGGUGCUUU*U*U*U*U (SEQ ID NO: 101) 2225′-7x(2′OMe,3′PACE)_plus3 CLTA1mg ON1- G _(o)*A _(o)*G_(o)*A*G*U*C*CUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUA  116NC_overhg_CLTA1_3′- targetACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU  4x(2′OMe,3′PACE)GGCACCGAGUCGGUGCUUU*U*U*U*U (SEQ ID NO: 101) 2235′-7x(2,OMe,3′PACE)_plus3  CLTA1mg ON1- G _(o)*A _(o)*G_(o)*A*G*U*C*CUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUA  116NC_overhg_CLTA1_3′- targetACAGCAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU  4x(2′OMe,3′PACE)GGCACCGAGUCGGUGCUUU*U*U*U*U (SEQ ID NO: 101) 224 CLTA1_2′OMe, CLTA1 ON1-AGUCCUCAUCUCCCUCAAGC*GUUUAAGAGCUAUGCUGGUAACAGCAUAGC  113 3′PACE +20 sgRNA target AAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 102 225 CLTA1_2′OMe, CLTA1mg ON1-AGUCCUCAUCUCCCUCAAGC*GUUUAAGAGCUAUGCUGGUAACAGCAUAGC  113 3′PACE +20 sgRNA target AAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 102) 226 CLTA1_2′OMe, CLTA1mg ON1-AGUCCUCAUCUCCCUCAAGC*GUUUAAGAGCUAUGCUGGUAACAGCAUAGC  113 3′PACE +20 sgRNA target AAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 102) 227 CLTA1_2′OMePACE + 19 sgRNA CLTA1 ON1-AGUCCUCAUCUCCCUCAAG*CGUUUAAGAGCUAUGCUGGUAACAGCAUAGC  113 targetAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 103) 228 CLTA1_2′OMePACE + 19 sgRNACLTA1mg ON1- AGUCCUCAUCUCCCUCAAG*CGUUUAAGAGCUAUGCUGGUAACAGCAUAGC  113target AAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 103) 229 CLTA1_2′OMePACE + 19 sgRNACLTA1mg ON1- AGUCCUCAUCUCCCUCAAG*CGUUUAAGAGCUAUGCUGGUAACAGCAUAGC  113target AAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 103) 230 CLTA1_2′OMePACE + 19 sgRNACLTA1mg OFF1- AGUCCUCAUCUCCCUCAAG*CGUUUAAGAGCUAUGCUGGUAACAGCAUAGC  113target AAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 103) 231 CLTA1_2′OMePACE + 19 sgRNACLTA1mg OFF3- AGUCCUCAUCUCCCUCAAG*CGUUUAAGAGCUAUGCUGGUAACAGCAUAGC  113target AAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 103) 232 CLTA1_2′OMePACE + 18 sgRNA CLTA1 ON1-AGUCCUCAUCUCCCUCAA*GCGUUUAAGAGCUAUGCUGGUAACAGCAUAGC  113 targetAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 104) 233 CLTA1_2′OMePACE + 18 sgRNACLTA1mg ON1- AGUCCUCAUCUCCCUCAA*GCGUUUAAGAGCUAUGCUGGUAACAGCAUAGC  113target AAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 104) 234 CLTA1_2′OMePACE + 18 sgRNACLTA1mg ON1- AGUCCUCAUCUCCCUCAA*GCGUUUAAGAGCUAUGCUGGUAACAGCAUAGC  113target AAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 104) 235 CLTA1_2′OMePACE + 17 sgRNA CLTA1 ON1-AGUCCUCAUCUCCCUCA*AGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGC  113 targetAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 105) 236 CLTA1_2′OMePACE + 17 sgRNACLTA1mg ON1- AGUCCUCAUCUCCCUCA*AGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGC  113target AAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 105) 237 CLTA1_2′OMePACE + 17 sgRNACLTA1mg ON1- AGUCCUCAUCUCCCUCA*AGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGC  113target AAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 105) 238 CLTA1_2′OMePACE + CLTA1 ON1-AGUCCUCAUCUCCCUCA*A*GCGUUUAAGAGCUAUGCUGGUAACAGCAUAG  113 17,18 sgRNAtarget CAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUUUUU (SEQ ID NO: 106) 239 CLTA1_2′OMePACE + CLTA1mg ON1-AGUCCUCAUCUCCCUCA*A*GCGUUUAAGAGCUAUGCUGGUAACAGCAUAG  113 17,18 sgRNAtarget CAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUUUUU (SEQ ID NO: 106) 240 CLTA1_2′OMePACE + CLTA1mg ON1-AGUCCUCAUCUCCCUCA*A*GCGUUUAAGAGCUAUGCUGGUAACAGCAUAG  113 17,18 sgRNAtarget CAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGCUUUUUUU (SEQ ID NO: 106)2′OMethyl,3′Phosphorothioate-modified sgRNA 241 CLTA1_5′,3′- CLTA1 ON1-A s G s U sCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAG  1133x(2′OMe, 3′P(S)) targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG  UCGGUGCUUU U s U s UsU (SEQ ID NO: 107) 242 CLTA1_5′,3′- CLTA1mg ON1- A s G s UsCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAG  113 3x(2′OMe, 3′P(S))target CAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG  UCGGUGCUUU Us U s U sU (SEQ ID NO: 107) 243 CLTA1_5′,3′- CLTA1mg ON1- A s G s UsCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAG  113 3x(2′OMe, 3′P(S))target CAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG  UCGGUGCUUU Us U s U sU (SEQ ID NO: 107) 244 CLTA1_5′,3′- CLTA1mg OFF1- A s G s UsCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAG  113 3x(2′OMe, 3′P(S))target CAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG  UCGGUGCUUU Us U s U sU (SEQ ID NO: 107) 245 CLTA1_5′,3′- CLTA1mg OFF3- A s G s UsCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAG  113 3x(2′OMe, 3′P(S))target CAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG  UCGGUGCUUU Us U s U sU (SEQ ID NO: 107) 246 CLTA4_5′,3′- CLTA4 G s C s AsGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAUA  113 3x(2′OMe, 3′P(S))target GCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGA  GUCGGUGCUUU Us U s U sU (SEQ ID NO: 107) 247 CLTA4 5′,3′- CLTA4  G s C s AsGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAUA  113 3x(2′OMe, 3′P(S))target GCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGA  GUCGGUGCUUU Us U s U sU (SEQ ID NO: 107) 248 CLTA4 5′,3′- CLTA4mg ON- G s C s AsGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAUA  113 3x(2′OMe, 3′P(S))target GCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGA  GUCGGUGCUUU Us U s U sU (SEQ ID NO: 107) 249 CLTA4 5′,3′- CLTA4mg ON- G s C s AsGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAUA  113 3x(2′OMe, 3′P(S))target GCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGA  GUCGGUGCUUU Us U s U sU (SEQ ID NO: 107) 250 CLTA4 5′,3′- CLTA4mg ON- G s C s AsGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAUA  113 3X(2′OMe, 3′P(S))target GCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGA  GUCGGUGCUUU Us U s U sU (SEQ ID NO: 107) 251 CLTA4 5′,3′- CLTA4mg OFF5- G s C s AsGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAUA  113 3x(2′OMe, 3′P(S))target GCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGA  GUCGGUGCUUU Us U s U sU (SEQ ID NO: 107) 252 CLTA4_5′- CLTA4mg ON- G s C s AsGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAUA  1133x(2′OMe, 3′P(S)),3′- targetGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGA  5x(2′OMe, 3′P(S))GUCGGUGCU U s U s U s U s U sU (SEQ ID NO: 108) 253 CLTA4_5′-CLTA4mg ON- G s C s A sGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAUA 113 3x(2′OMe, 3′P(S)),3′- targetGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGA  5x(2′OMe, 3′P(S))GUCGGUGCU U s U s U s U s U sU (SEQ ID NO: 108) 254 CLTA4_5′-CLTA4mg ON- G s C s A sGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAUA 113 3x(2′OMe, 3′P(S)),3′- targetGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGA  5x(2′OMe, 3′P(S))GUCGGUGCU U s U s U s U s U sU (SEQ ID NO: 108) 255 CLTA4 5′,3′-CLTA4mg ON- G s C s A s G s AsUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAU  113 5x(2′OMe, 3′P(S))target AGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG  AGUCGGUGCU Us U s U s U s U sU (SEQ ID NO: 109) 256 CLTA4 5′,3′- CLTA4mg ON- G s C sA s G s A sUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAU  1135x(2′OMe, 3′P(S)) targetAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG  AGUCGGUGCU U s U s Us U s U sU (SEQ ID NO: 109) 257 CLTA4 5′,3′- CLTA4mg OFF5- G s C s A s Gs A sUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAU  113 5x(2′OMe, 3′P(S))target AGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG  AGUCGGUGCU Us U s U s U s U sU (SEQ ID NO: 109)2′OMethyl,3′PhosphorothioPACE-modified sgRNA 258 CLTA1_5′,3′- CLTA1 ON1-A *s G *s U *sCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCA  1133x(2′OMe, 3′thioPACE) targetUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC  GAGUCGGUGCUUU U *s U*s U *sU (SEQ ID NO: 110) 259 CLTA1_5′,3′- CLTA1mg ON1- A *s G *s U*sCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCA  113 3x(2′OMe, 3′thioPACE)target UAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC  GAGUCGGUGCUUUU *s U *s U *sU (SEQ ID NO: 110) 260 CLTA1_5′,3′- CLTA1mg ON1- A *s G *sU *sCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCA  1133x(2′OMe, 3′thioPACE) targetUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC  GAGUCGGUGCUUU U *s U*s U *sU (SEQ ID NO: 110) 261 CLTA1_5′,3′- CLTA1mg OFF1- A *s G *s U*sCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCA  113 3x(2′OMe, 3′thioPACE)target UAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC  GAGUCGGUGCUUUU *s U *s U *sU (SEQ ID NO: 110) 262 CLTA1_5′,3′- CLTA1mg OFF3- A *s G*s U *sCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCA  1133x(2′OMe, 3′thioPACE) targetUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC  GAGUCGGUGCUUU U *s U*s U *sU (SEQ ID NO: 110) 263 CLTA1_5′,3′- CLTA1mg ON1- A*sGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAG  1131x(2′OMe, 3′thioPACE) targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG  UCGGUGCUUUUU U*sU (SEQ ID NO: 111) 264 CLTA1_5′,3′- CLTA1mg ON1- A*sGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAG  1131x(2′OMe, 3′thioPACE) targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG  UCGGUGCUUUUU U*sU (SEQ ID NO: 111) 265 CLTA1_5′,3′- CLTA1mg OFF1- A*sGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAG  1131x(2′OMe, 3′thioPACE) targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG  UCGGUGCUUUUU U*sU (SEQ ID NO: 111) 266 CLTA1_5′,3′- CLTA1mg OFF3- A*sGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAG  1131x(2′OMe, 3′thioPACE) targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG  UCGGUGCUUUUU U*sU (SEO ID NO: 111) 267 CLTA1_5′,3′-3x(2′OMe, CLTA1 ON1- A *s G *s U*sCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGC 75 3′thioPACE)_75mer targetAUAGCAAGUUUAAAUAAGGCUAGUCC G *s U *s U *sU (SEQ ID NO: 112) 268CLTA1_5′,3′-1x(2′OMe, CLTA1mg ON1- A*sGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAG  74 3′thioPACE)_74mertarget CAAGUUUAAAUAAGGCUAGUCCG U *sU (SEQ ID NO: 112) 269CLTA1_5′,3′-1x(2′OMe, CLTA1mg ON1- A*sGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAG  75 3′thioPACE)_75mertarget CAAGUUUAAAUAAGGCUAGUCCGU U *sA (SEQ ID NO 112) 270CLTA1_5′,3′-1x(2′OMe, CLTA1mg ON1- A*sGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAG  77 3′thioPACE)_77mertarget CAAGUUUAAAUAAGGCUAGUCCGUUA U *sC (SEQ ID NO: 113) 271CLTA1_5′,3′-1x(2′OMe, CLTA1mg ON1- G*sAGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUA  78 3′thioPACE)_77mertarget GCAAGUUUAAAUAAGGCUAGUCCGUUA U *sC (SEQ ID NO: 114) 272CLTA4_5′,3′- CLTA4 ON- G *s C *s A*sGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCA  113 3x(2′OMe, 3′thioPACE)target UAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC  GAGUCGGUGCUUUU *s U *s U *sU (SEQ ID NO: 115) 273 CLTA4_5′,3′- CLTA4 ON- G *s C *s A*sGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCA  113 3x(2′OMe, 3′thioPACE)target UAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC  GAGUCGGUGCUUUU *s U *s U *sU (SEQ ID NO: 115) 274 CLTA4_5′,3′- CLTA4 ON- G *s C *s A*sGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCA  113 3x(2′OMe, 3′thioPACE)target UAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC  GAGUCGGUGCUUUU *s U *s U *sU (SEQ ID NO: 115) 275 CLTA4_5′,3′- CLTA4mg OFF5- G *s C*s A *sGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCA  1133x(2′OMe, 3′thioPACE) targetUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC  GAGUCGGUGCUUU U *s U*s U *sU (SEQ ID NO: 115) 276 CLTA4_5′,3′- CLTA4mg ON- G*sCAGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAUAG  1131x(2′OMe, 3′thioPACE) targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG  UCGGUGCUUUUU U*sU (SEQ ID NO: 116) 277 CLTA4_5′,3′- CLTA4mg ON- G*sCAGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAUAG  1131x(2′OMe, 3′thioPACE) targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG  UCGGUGCUUUUU U*sU (SEQ ID NO: 116) 278 CLTA4_5′,3′- CLTA4mg ON- G*sCAGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAUAG  1131x(2′OMe, 3′thioPACE) targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG  UCGGUGCUUUUU U*sU (SEQ ID NO: 116) 279 CLTA4_5′,3′- CLTA4mg OFF5- G*sCAGAUGUAGUGUUUCCACAGUUUAAGAGCUAUGCUGGAAACAGCAUAG  1131x(2′OMe, 3′thioPACE) targetCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG  UCGGUGCUUUUU U*sU (SEQ ID NO: 116)2-aminoA-modified sgRNA (including unmodified controls) 280 EN1EN1mg ON- GAUGUUGUCGAUGAAAAAGUGUUUAAGAGCUAUGCUGGUAACAGCAUAGC  113 targetAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 117) 281 EN1 EN1mg OFF-GAUGUUGUCGAUGAAAAAGUGUUUAAGAGCUAUGCUGGUAACAGCAUAGC  113 targetAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 117) 282 EN1_2aminoA + 16 EN1mg ON-GAUGUUGUCGAUGAA(2aA)AAGUGUUUAAGAGCUAUGCUGGUAACAGCAU  113 targetAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 118) 283 EN1_2aminoA + 16 EN1mg OFF-GAUGUUGUCGAUGAA(2aA)AAGUGUUUAAGAGCUAUGCUGGUAACAGCAU  113 targetAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG AGUCGGUGCUUUUUUU (SEQ ID NO: 118) 284 PCDHA4 PCDHA4mg ON-GAUUUAGACGAAGGAUUGAAGUUUAAGAGCUAUGCUGGUAACAGCAUAGC  113 targetAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 119) 285 PCDHA4 PCDHA4mg OFF-GAUUUAGACGAAGGAUUGAAGUUUAAGAGCUAUGCUGGUAACAGCAUAGC  113 targetAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU CGGUGCUUUUUUU (SEQ ID NO: 119) 286 PCDHA4_2aminoA + 15 PCDHA4mg ON-GAUUUAGACGAAGG(2aA)UUGAAGUUUAAGAGCUAUGCUGGUAACAGCAU  113 targetAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG AGUCGGUGCUUUUUUU (SEQ ID NO: 120) 287 PCDHA4_2aminoA + 15 PCDHA4mg OFF-GAUUUAGACGAAGG(2aA)UUGAAGUUUAAGAGCUAUGCUGGUAACAGCAU  113 targetAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG AGUCGGUGCUUUUUUU (SEQ ID NO: 120) 5-methylU-modified sgRNA 288CLTA4_21x(5-MeU) CLTA4mg ON-GCAGA(5mU)G(5mU)AG(5mU)G(5mU)(5mU)(5mU)CCACAGUUUAAG 113 targetAGC(5mU)A(5mU)GC(5mU)GG(5mU)AACAGCA(5mU)AGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAAC(5mU)(5mU)GAAAAAG(5mU)GGCACCGAGUCGG(5mU)GC(5mU)(5mU)(5mU)(5mU)(5mU)(5mU)U (SEQ ID NO: 121) 289CLTA4_21x(5-MeU) CLTA4mg OFF5-GCAGA(5mU)G(5mU)AG(5mU)G(5mU)(5mU)(5mU)CCACAGUUUAAG 113 targetAGC(5mU)A(5mU)GC(5mU)GG(5mU)AACAGCA(5mU)AGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAAC(5mU)(5mU)GAAAAAG(5mU)GGCACCGAGUCGG(5mU)GC(5mU)(5mU)(5mU)(5mU)(5mU)(5mU)U  (SEQ ID NO: 121)Z base-modified sgRNA 290 CLTA1_ZZ_70,71 CLTA1 ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAGGCUAGUZZGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUU (SEQ ID NO: 122) 291 CLTA1 ZZ_95,96 CLTA1 ON1-AGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUAGCA  113 targetAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCAZZGAGUC GGUGCUUUUUUU (SEQ ID NO: 122) sgRNA modified to disfavor misfolding 292CLTA1_opti_short_5′,3′- CLTA1mg ON1- A*sGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAGUAAUAGCAAGUUUAA 100 1x(2′OMe,  targetAUAAGG U UA A UCCGUUAUCAACAAGAAAUUGUGGCACCGAGUCGGUGCUU3′thioPACE)_2′OMe_54,57 U *sU (SEQ ID NO: 123) 293CLTA1_opti_short_5′,3′- CLTA1mg ON1- A*sGUCCUCAUCUCCCUCAAGCGUUUAAGAGCUAUGCUGGUAACAGCAUA 113 1x(2′OMe,  targetGCAAGUUUAAAUAAGG U UA A UCCGUUAUCAACAAGAAAUUGUGGCACCGA3′thioPACE)_2′OMe_64,67 GUCGGUGCUUUUU U *sU (SEQ ID NO: 124) N = 2′OMe N= 2′deoxy Ns = 3′P(S) N* = 3′-PACE N*s = 3′-thioPACE N* = 2′OMe,3′-PACEN*s = 2′OMe,3′-thioPACE Ns = 2′OMe, 3′P(S) N_(o) = 5′-overhang (5′ tothe 20-nt guide sequence); “NC” means the overhang is not complementaryto protospacer-adjacent sequence (2sU) = 2-thioU (2aA) = 2-aminoA (5mU)= 5-methylU Z = Zbase IntFl = Fluorophore incorporated at an internalposition in the RNA sequence

The DNA target constructs in Table 3 had the following sequences:

CLTA1 ON1- AGAATTTAACTGTGGTCACATTTGCTTTATCGACTGGCTTCATCTCACAGCTCATCtarget: TTACGCAAGTTCGATGAGTATGCCAGTCACTTTCAATTTGGTTGAATGTTCCCGTGACATGCGAGTTCTGTCGACCATGTGCCGCGGATTGAATTCCTCAAGGGTGGTGATAGATGCTACGGTGGTGATGCGCATGCGCTCAGTCCTCATCTCCCTCAAGCAGGCCCCGCTGGTGGGTCGGAGTCCCTAGTGAAGCCACCAATATAGTGGTCGTGTCAAGCAACTGTCCACGCTCCACCCTCGAGGTCGTAACATAAACGTACTAAGGCACGAGTAAACAAGATCGATAGCAAGAACATGGTATAGACTGACGGAGAGCTCGCCATTAGTCTGA (SEQ ID NO: 10)CLTA1 OFF1- AGAATTTAACTGTGGTCACATTTGCTTTATCGACTGGCTTCATCTCACAGCTCATCtarget: TTACGCAAGTTCGATGAGTATGCCAGTCACTTTCAATTTGGTTGAATGTTCCCGTGACATGCGAGTTCTGTCGACCATGTGCCGCGGATTGAATTCCTCAAGGGTGGTGATAGATGCTACGGTGGTGATGCGTATGCACTCAGTCCTCAACTCCCTCAAGCAGGCGACCCCTGGGGGTCGGAGTCCCTAGTGAAGCCACCAATATAGTGGTCGTGTCAAGCAACTGTCCACGCTCCACCCTCGAGGTCGTAACATAAACGTACTAAGGCACGAGTAAACAAGATCGATAGCAAGAACATGGTATAGACTGACGGAGAGCTCGCCATTAGTCTGA (SEQ ID NO: 11)CLTA1 OFF2- AGAATTTAACTGTGGTCACATTTGCTTTATCGACTGGCTTCATCTCACAGCTCATCtarget: TTACGCAAGTTCGATGAGTATGCCAGTCACTTTCAATTTGGTTGAATGTTCCCGTGACATGCGAGTTCTGTCGACCATGTGCCGCGGATTGAATTCCTCAAGGGTGGTGATAGATGCTACGGTGGTGATGCAATAAATTTCAGCCCTCATTTCCCTCAAGCAGGGGTTACTTTAGGGTCGGAGTCCCTAGTGAAGCCACCAATATAGTGGTCGTGTCAAGCAACTGTCCACGCTCCACCCTCGAGGTCGTAACATAAACGTACTAAGGCACGAGTAAACAAGATCGATAGCAAGAACATGGTATAGACTGACGGAGAGCTCGCCATTAGTCTGA (SEQ ID NO: 12)CLTA1 OFF3- AGAATTTAACTGTGGTCACATTTGCTTTATCGACTGGCTTCATCTCACAGCTCATCtarget: TTACGCAAGTTCGATGAGTATGCCAGTCACTTTCAATTTGGTTGAATGTTCCCGTGACATGCGAGTTCTGTCGACCATGTGCCGCGGATTGAATTCCTCAAGGGTGGTGATAGATGCTACGGTGGTGATGCTCTCCAGCCCACTCCTCATCCCCCTCAAGCCGGTCCCAGGCTGGGGTCGGAGTCCCTAGTGAAGCCACCAATATAGTGGTCGTGTCAAGCAACTGTCCACGCTCCACCCTCGAGGTCGTAACATAAACGTACTAAGGCACGAGTAAACAAGATCGATAGCAAGAACATGGTATAGACTGACGGAGAGCTCGCCATTAGTCTGA (SEQ ID NO: 13)CLTA1mg ON1- GCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGtarget: GCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACGATCGATGCGGCCTCGCAGGCCAAAGATGTCTCCCGCATGCGCTCAGTCCTCATCTCCCTCAAGCAGGCCCTGCTGGTGCACTGAAGAGCCACCCTGTGCGCGTGATATGCAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGC (SEQ ID NO: 14) CLTA1mg OFF1-GCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGG target:GCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACGATCGATGCGGCCTCGCAGGGCAAAGAGGTCTCCTGTATGCACTCAGTCCTCAACTCCCTCAAGCAGGCGACCCTTGGTGCACTGACAAACCGCTCCTGCGCGTGATATGCAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGC (SEQ ID NO: 15) CLTA1mg_OFF3GCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGG target:GCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACGATCGATGCGGCCTCAGGAGAGGGAGCCATGCTCATCTCCAGCCCACTCCTCATCCCCCTCAAGCCGGTCCCAGGCTGAGAGGCTAAAGCTTGTCTTTGCGCGTGATATGCAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGC (SEQ ID NO: 16) CLTA4 ON-GCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGA target:GTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACtGaGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCGCGCAATTAACCCTCACTAAAGGGAACAAAAGCTGGGTACCGGGCCCCCCCTCGACACCAGTTGCATTCGATTCCTGTTTGTAATTGTCCAATTCCTGCAGCCCGGGGGATCGGCAGATGTAGTGTTTCCACAGGGGATCCACTAGTTCTAGAGCGGCCGCCACCGCGGTGGAGCTCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGAC (SEQ ID NO: 17) CLTA4mg ON-GCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGG target:GCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACGATCGATGCGGCCTCAAGAGCTTCACTGAGTAGGATTAAGATATTGCAGATGTAGTGTTTCCACAGGGTGGCTCTTCAGTGCACCAGCGGAACCTGCTGCGCGTGATATGCAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGC (SEQ ID NO: 18) CLTA4mg OFF5-GCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGG target:GCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACGATCGATGCGGCCTCTCTGAATAGAGTTGGGAAGAGATGCATACAACATATGTAGTATTTCCACAGGGAATACAATGGACAAATGACCTCAAGAGCAGGCGCGTGATATGCAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGC (SEQ ID NO: 19) IL2RGmg_ONGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGG target:GCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACGATCGATGCGGCCTCGGGCAGCTGCAGGAATAAGAGGGATGTGAATGGTAATGATGGCTTCAACATGGCGCTTGCTCTTCATTCCCTGGGTGTAGTCTGCGCGTGATATGCAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGC (SEQ ID NO: 20) EN1mg_ONGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGG target:GCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACGATCGATGCGGCCTCCTCCTTACTGCAGCCGAAGTCCGGCCTCAGGATGTTGTCGATGAAAAAGTTGGTGGTGCGGTGCAGCTGGGCCGCTGGCTGCGGCGCGTGATATGCAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGC (SEQ ID NO: 21) EN1mg_OFFGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGG target:GCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACGATCGATGCGGCCTCGTCCTTTCGCCGGCCGAACTCGGGCCGCAGGATGTTGTCGATGAAGAAGTTGGTGATGCGGTGCGGGTGCTGGTGGTTGCCGGGCGCGTGATATGCAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGC (SEQ ID NO: 22) PCDHA4mg_ONGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGG target:GCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACGATCGATGCGGCCTCGGAACATTGGTAATTAAACTTAACGCCTCAGATTTAGACGAAGGATTGAATGGGGACATTGTTTATTCATTCTCGAATGATACGCGCGTGATATGCAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGC (SEQ ID NO: 23) PCDHA4mg_OFFGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGG target:GCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACGATCGATGCGGCCTCGGAACGCTGGTGATTCATCCCAATGCCTCAGATTTAGACGAAGGCTTGAATGGGGATATTATTTACTCCTTCTCCAGTGATGTGCGCGTGATATGCAGCTCCAGCTTTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGC (SEQ ID NO: 24)

In a 20-uL reaction volume, 50 fmoles of linearized DNA target in thepresence of 50 nM sgRNA, 39 nM recombinant purified Cas9 protein (S.pyogenes; Agilent) and 10 mM or 0.8 mM MgCl₂ at pH 7.6 was incubated at37° C. for 30 min. Upon completion, 0.5 uL of RNace It (Agilent) wasadded, and incubation was continued at 37° C. for 5 min and then at 70°C. for 15 min. Subsequently 0.5 μL of Proteinase K (Mol. Bio. grade,NEB) was added and incubated at 37° C. for 15 min. Aliquots were loadedinto a DNA 1000 or DNA 7500 LabChip and were analyzed on a Bioanalyzer2200, or alternatively were loaded into a Genomic DNA ScreenTape andwere analyzed on a TapeStation. The workup steps served to release Cas9from binding of target DNA, which was assayed for cleavage. Cleavageyields were calculated by the formula: a/(a+b)×100 where a is the sum ofthe band intensities of the two cleavage products and b is the remaininguncleaved DNA if present. A cleavage percentage of 100% means that allof the target DNA construct was cleaved.

A series of guide RNAs were chemically synthesized. The guide RNAoligomers were synthesized on an ABI 394 Synthesizer (Life Technologies,Carlsbad, Calif., USA) using 2′-O-thionocarbamate-protected nucleosidephosphoramidites according to procedures described in Dellinger et al.(2011) J. Am. Chem. Soc., 133, 11540-56. 2′-O-methyl phosphoramiditeswere incorporated into RNA oligomers under the same conditions as the2′-O-thionocarbamate protected phosphoramidites. The2′-O-methyl-3′-O-(diisopropylamino)phosphinoaceticacid-1,1-dimethylcyanoethyl ester-5′-O-dimethoxytrityl nucleosides usedfor synthesis of thiophosphonoacetate (thioPACE)-modified RNAs weresynthesized essentially according to published methods. See Dellinger etal. (2003) J. Am. Chem. Soc., 125, 940-50; and Threlfall et al. (2012)Org. Biomol. Chem., 10, 746-54. For phosphorothioate-containingoligomers, the iodine oxidation step after the coupling reaction wasreplaced by a sulfurization step using a 0.05 M solution of3-((N,N-dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-5-thione ina pyridine-acetonitrile (3:2) mixture for 6 min.

All the oligonucleotides were purified using reversed-phasehigh-performance liquid chromatography (HPLC) and analyzed by liquidchromatography-mass spectrometry (LC-MS) using an Agilent 1290 Infinityseries LC system coupled to an Agilent 6520 Q-TOF (time-of-flight) massspectrometer (Agilent Technologies, Santa Clara, Calif., USA). Theyields for the synthesis and purification of the-sgRNAs were estimatedusing deconvolution of mass spectra obtained from LC-MS-derived totalion chromatograms. The chemical synthesis of the 100-mer sgRNAstypically yielded 25-35% full-length product from a nominal 1 micromolescale synthesis. Reversed-phase HPLC purification using ion pairingbuffer conditions typically gave 20% yield from the crude product withan estimated purity of the final sgRNA in the range of 90% to 95%.

The results are shown in Table 4. “% Target cleaved” indicates thepercentage of the target DNA construct which was cleaved. Experimentswere run with and without addition of a molar excess of targetlesscompetitor DNA (tcDNA) which potentially competes with the target DNA,so the potential impact of the added nonspecific DNA upon the assaycould be seen.

TABLE 4 % Cleaved % CV Entry [Mg²⁺] % Target vs. CON- # (mM) tcDNAcleaved % CV CONTROL TROL 2-piece dual-guide scaffold Unmodifieddual-guide RNA (dgRNA) 1 0.8 N 99% — 2 0.8 Y 99% 5% 3 0.8 N 96% — 4 0.8Y 100%  5% 5 0.8 N 96% — 6 0.8 Y  0% 5% 7 0.8 N 99% — 8 0.8 Y 100%  5% 910 N 94%, 93% — 10 0.8 Y 88% — Fluorophore-coupled dgRNA 11 10 N 92%,93% — 94%, 93% — 2′OMethyl-modified dgRNA 12 0.8 Y 87% — 88% —2′OMethyl,3′Phosphorothioate-modified dgRNA 13 0.8 Y 87% — 88% —2′OMethyl,3′PhosphorothioPACE-modified dgRNA 14 0.8 Y 89% — 88% — 15 0.8Y 86% — 88% — 2-thioU-modified dgRNA 16 0.8 N 96% — 99% — 17 0.8 Y 95%5% 99% 5% 18 0.8 N 95% — 96% — 19 0.8 Y 100%  5% 100%  5% 20 0.8 N 97% —96% — 21 0.8 Y  0% 5%  0% 5% 22 0.8 N 98% — 99% — 23 0.8 Y 99% 5% 100% 5% 24 0.8 N 94% — 99% — 25 0.8 Y 83% 5% 99% 5% 26 0.8 N 93% — 96% — 270.8 Y 94% 5% 100%  5% 28 0.8 N 90% — 96% — 29 0.8 Y  0% 5%  0% 5% 30 0.8N 95% — 99% — 31 0.8 Y 94% 5% 100%  5% 32 0.8 N 92% — 99% — 33 0.8 Y 84%5% 99% 5% 34 0.8 N 90% — 96% — 35 0.8 Y 94% 5% 100%  5% 36 0.8 N 70% —96% — 37 0.8 Y  0% 5%  0% 5% 38 0.8 N 96% — 99% — 39 0.8 Y 59% 5% 100% 5% Single-guide scaffold Unmodified single-guide RNA (sgRNA) 40 10 N 93%— 41 10 N 94% — 42 10 N 94% — 43 10 N 92% — 44 10 N 90%, 92% — 45 10 N92% — 46 10 N 93% — 47 0.8 N 86% — 48 0.8 N 87% — 49 0.8 Y 87% — 50 0.8N 82% — 51 0.8 N 92% — 52 10 N 60% — 53 0.8 N 90% — 54 0.8 N 90% — 550.8 Y 79% — 56 0.8 N 79% — 57 0.8 N 94% — 58 10 N 73% — 59 0.8 N 84% —60 0.8 Y ≧85%   — 61 0.8 Y 89% — 62 0.8 N 87%, 82% — 63 0.8 N 23%, 22% —64 0.8 N 78% — 87% — 65 0.8 Y 76% — 87% — 66 0.8 N 65% — 87% — 67 0.8 N81% — 87% — 68 0.8 N 85% — 87% — 69 0.8 Y 71% — 87% — 70 0.8 N 32% — 87%— 71 0.8 N 84% — 87% — 72 0.8 N 91% — 87% — 73 0.8 Y 79% — 87% — 74 0.8N 88% — 87% — 75 0.8 N 93% — 87% — 76 0.8 N 87% — 87% — 77 0.8 Y 79% —87% — 78 0.8 N 89% — 87% — 79 0.8 N 88% — 87% — 80 0.8 N  3% — 86% — 810.8 N  5% — 86% — 82 0.8 N 89% — 86% — 83 0.8 N 68% — 87% — 84 0.8 Y 50%— 87% — 85 0.8 N 69% — 87% — 86 0.8 N 69% — 87% — 87 0.8 N 76% — 87% —88 0.8 Y 42% — 87% — 89 0.8 N 72% — 87% — 90 0.8 N 78% — 87% — 91 0.8 N85% — 87% — 92 0.8 Y 51% — 87% — 93 0.8 N 82% — 87% — 94 0.8 ″ 83% — 87%— DMT-modified sgRNA 95 10 N 93% — 92% — 96 10 N 93% — 92% —Fluorophore-modified sgRNA 97 10 N 91%, 91% — 90%, 92% — 98 0.8 N 86% —87% — 99 0.8 Y 77% — 87% — 100 0.8 N 87% — 87% — 101 0.8 N 86% — 87% —102 0.8 N 91% — 87% — 103 0.8 Y 82% — 87% — 104 0.8 N 90% — 87% — 1050.8 N 92% — 87% — 106 0.8 N 91% — 87% — 107 0.8 Y 82% — 87% — 108 0.8 N90% — 87% — 109 0.8 N 91% — 87% — 110 0.8 N 92% — 87% — 111 0.8 Y 84% —87% — 112 0.8 N 92% — 87% — 113 0.8 N 89% — 87% — 114 0.8 N 84%, 84% —87%, 82% — 115 0.8 N 12%, 6%  — 23%, 22% — 116 0.8 N 93%, 90% — 87%, 82%— 117 0.8 N 8%, 9% — 23%, 22% — 3′Phosphorothioate-modified sgRNA 118 10N 95% — 90%, 92% — 119 10 N 94% — 90%, 92% — 120 10 N 97% — 90%, 92% —121 10 N 94% — 90%, 92% — 2′OMethyl-modified sgRNA 122 10 N 91% — 94% —123 10 N 92% — 93% — 124 0.8 N 86% — 87% — 125 ″ Y 77% — 87% — 126 ″ N85% — 87% — 127 0.8 N 88% — 87% — 128 10 N 92% — 94% — 129 0.8 N 83% —87% — 130 0.8 Y 78% — 87% — 131 0.8 N 83% — 87% — 132 0.8 N 85% — 87% —133 10 N 92% — 94% — 134 0.8 N 86% — 87% — 135 0.8 Y 78% — 87% — 136 0.8N 83% — 87% — 137 0.8 N 88% — 87% — 138 10 N 91% — 94% — 139 0.8 N 84% —87% — 140 0.8 Y 81% — 87% — 141 0.8 N 83% — 87% — 142 0.8 N 87% — 87% —143 10 N 89% — 92% — 144 0.8 N 91%, 88% — 87%, 82% — 145 0.8 N 24%, 25%— 23%, 22% — 146 10 N 93%, 92% — 90%, 92% — 147 0.8 N 22% — 87% — 1480.8 Y  3% — 87% — 149 0.8 N 12% — 87% — 150 0.8 N  5% — 87% — 151 10 N0%, 0% — 90%, 92% — 152 10 N 0%, 0% — 90%, 92% — 153 0.8 N 85% — 86% —154 0.8 N 87% — 86% — 155 0.8 N 89% — 87% — 156 0.8 Y 78% — 87% — 1570.8 N 84% — 87% — 158 0.8 N 93% — 87% — 159 0.8 N 90% — 86% — 160 0.8 N90% — 87% — 161 0.8 Y 86% — 87% — 162 0.8 N 90% — 87% — 163 0.8 N 91% —87% — 164 0.8 N 92% — 90% — 165 0.8 N 89% — 87% — 166 0.8 Y 80% — 87% —167 0.8 N 90% — 87% — 168 0.8 N 94% — 87% — 169 0.8 N 90% — 84% — 1700.8 Y ≧85%   — ≧85%   — 171 0.8 N  7% — 84% — 172 0.8 Y  0% — ≧85%   —173 10 N 15% — 73% — 174 0.8 N 85% — 84% — 175 0.8 Y 75% — ≧85%   — 17610 N 86% — 73% — 177 0.8 ″  0% — 84% — 178 0.8 Y  0% — ≧85%   — 179 10 N15% — 73% — 2′Deoxy-modified sgRNA 180 10 N 27%, 19% — 90%, 92% — 181 10N 0%, 0% — 90%, 92% — 182 10 N 0%, 0% — 90%, 92% —2′Deoxy,3′PACE-modified sgRNA 183 0.8 N 72%, 77% — 87%, 82% — 184 0.8 N8%, 9% — 23%, 22% — 2′OMethyl,3′PACE-modified sgRNA 185 0.8 N 82% — 87%— 186 0.8 Y 72% — 87% — 187 10 Y 95% — 93% — 188 10 Y 95% — 94% — 1890.8 Y 91% — 87% — 190 0.8 Y 84% — 87% — 191 0.8 Y 85% — 87% — 192 0.8 Y77% — 87% — 193 10 Y 88% — 94% — 194 0.8 Y 70% — 87% — 195 0.8 Y 56% —87% — 196 0.8 Y 40% — 87% — 197 0.8 Y 23% — 87% — 198 10 Y 88% — 93% —199 10 Y 89% — 94% — 200 0.8 Y 84% — 87% — 201 0.8 Y 75% — 87% — 202 10Y 90% — 93% — 203 10 Y 90% — 94% — 204 0.8 Y 86% — 87% — 205 0.8 Y 82% —87% — 206 10 Y 88% — 93% — 207 0.8 Y 82% — 87% — 208 0.8 Y 78% — 87% —209 10 Y 77% — 93% — 210 0.8 Y 71% — 87% — 211 ″ Y 69% — ″ — 212 10 N80% — 93% — 213 0.8 N 56% — 87% — 214 0.8 Y 41% — ″ — 215 10 Y 78% — 93%— 216 0.8 Y 58% — 87% — 217 0.8 Y 44% — ″ — 218 10 Y 80% — 93% — 219 0.8Y 39% — 87% — 220 0.8 Y 13% — ″ — 221 10 Y 74% — 93% — 222 0.8 Y 36% —87% — 223 0.8 Y 19% — 87% — 224 10 Y 86% — 93% — 225 0.8 Y 84% — 87% —226 0.8 Y 80% — ″ — 227 10 Y 88% — 93% — 228 0.8 Y 83% — 87% — 229 0.8 Y82% — 87% — 230 0.8 N 80% — 87% — 231 0.8 N 84% — 87% — 237 10 N 88% —93% — 233 0.8 N 85% — 87% — 234 0.8 Y 73% — 87% — 235 10 Y 82% — 93% —236 0.8 Y 89% — 87% — 237 0.8 Y 76% — 87% — 238 10 Y 65% — 93% — 239 0.8Y 84% — 87% — 240 0.8 Y 56% — 87% —2′OMethyl,3′Phosphorothioate-modified sgRNA 241 10 N 92% — 92% — 242 0.8N 84% — 87% — 243 0.8 Y 88% — 87% — 244 0.8 N 85% — 87% — 245 0.8 N 91%— 87% — 246 0.8 N 91% — 84% — 247 0.8 Y ≧85%   — ≧85%   — 248 0.8 N 84%— 84% — 249 0.8 Y 90% — 89% — 250 0.8 N 90%, 87% — 87%, 82% — 251 0.8 N16%, 19% — 23%, 22% — 252 0.8 N 93% — 89% — 253 0.8 N 90%, 90% — 87%,82% — 254 0.8 N 17%, 22% — 23%, 22% — 255 0.8 N 93% — 89% — 256 0.8 N91%, 91% — 87%, 82% — 257 0.8 N 13%, 16% — 23%, 22% —2′OMethyl,3′PhosphorothioPACE-modified sgRNA 258 10 N 89% — 92% — 2590.8 N 84% — 87% — 260 0.8 Y 80% — 87% — 261 0.8 N 77% — 87% — 262 0.8 N83% — 87% — 263 0.8 N 92% — 87% — 264 0.8 Y 79% — 87% — 265 0.8 N 88% —87% — 266 0.8 N 94% — 87% — 267 10 N 74% — 93% — 268 0.8 N 11% — 86% —269 0.8 N 15% — ″ — 270 0.8 N 49% — ″ — 271 0.8 N 31% — ″ — 272 0.8 N91% — 84% — 273 0.8 Y 77% — ≧85%   — 274 0.8 N 90%, 91% — 87%, 82% — 2750.8 N 9%, 8% — 23%, 22% — 276 0.8 N 90% — 84% — 277 0.8 Y ≧85%   —≧85%   — 278 0.8 N 86%, 88% — 87%, 82% — 279 0.8 N 11%, 7%  — 23%, 22% —2-aminoA-modified-sgRNA (including unmodified controls) 280 0.8 Y 88%,88% — 281 0.8 Y 76%, 75% — 282 0.8 Y 87%, 91% — 88%, 88% — 283 0.8 Y90%, 90% — 76%, 75% — 284 0.8 Y 85%, 87% — 285 0.8 Y 88%, 88% — 286 0.8Y 93%, 96% — 85%, 87% — 287 0.8 Y 82%, 79% — 88%, 88% —5-methylU-modified sgRNA 288 0.8 N 86%, 83% — 87%, 82% — 289 0.8 N 11%,11% — 23%, 22% — Z base-modified sgRNA 290 10 N 19% — 92% — 291 10 N 93%— ″ — sgRNA modified to disfavor misfolding 292 0.8 N 93% — 90% — 2930.8 N 93% — 86% —

The results revealed that guide RNAs containing modifications atspecific positions were tolerated by active Cas protein and gRNA:Casprotein complexes, as the modifications did not prevent target-specificcleavage of the on-target polynucleotide. The modifications that weretested and found to be tolerated at specific positions include2-O-methylribonucleotide (=2′OMe), 2′-deoxyribonucleotide, racemicphosphorothioate internucleotide linkage, 3′-phosphonoacetate (=PACE),3-thiophosphonoacetate (=thioPACE), Z nucleotide, 2-thiouracil,2-aminoadenine, 5-methyluracil, 5-aminoallyluracil coupled to Cy5fluorophore, 2-(4-butylamidofluorescein)propane-1,3-diolbis(phosphodiester) linker, and combinations of these.

It is contemplated that the chemical modifications disclosed and testedherein, particularly at the tested positions (as listed in Tables 3 and4), will be tolerated at equivalent positions in a variety of guideRNAs.

As disclosed herein, chemically modified nucleotides were incorporatedinto guide RNAs in an effort to improve certain properties. Suchproperties include improved nuclease resistance of the guide RNA (alsoknown as improved stability), reduced off-target effects of a gRNA:)Casprotein complex (also known as improved specificity), improved efficacyof gRNA:Cas protein complex when cleaving, nicking or binding a targetpolynucleotide, improved transfection efficiency, and/or improvedorganelle localization.

The assay results in Tables 3 and 4 indicate that: (1) In guide RNAs,many positions can tolerate a variety of chemical modifications; (2) 5′and 3′ ends of guide RNAs will tolerate a wide variety of end protectingmodifications, and such modifications are useful to inhibitexonucleolytic RNA degradation; (3) 2-ThioU can be used to deteroff-target interactions involving G-U wobble pairings, therebyincreasing the specificity of guide pairing by inhibiting off-targethybridization interactions; (4) 5′ Extensions are generallywell-tolerated; (5) Surface exposed regions of the guide RNA (asinferred from published crystal structures) are tolerant of extensivelymodifying U's to 5-methylU's, which potentially make the modified RNAmore likely to elude immune responses such as stimulated by unmodifiedRNA; and (6) For RNA folding, G-C pairs are stronger and more stablethan A-U pairs. At least one guide RNA is tolerant of replacing some G-Cpairs with 2′-O-methylA-2′-O-methylU pairs that are more stablethermodynamically than unmodified A-U pairs.

More particularly, the present example shows that 2′-O-methylmodifications are tolerated at the 5′ and 3′ ends of dual-guide RNAs (asshown by entry 12 in Tables 3 and 4) and single-guide RNAs (entries143-146, 169-170), thus allowing end-protection to stabilize gRNAsagainst exonucleases. 2′-O-methyl modifications are tolerated at mostbut not all internal positions, thus allowing stabilization againstvarious nucleases including endonucleases (entries 146, 153-168,174-179). However, the present example also demonstrates that not everyposition in guide RNAs will tolerate 2′-O-methyls (as shown by entries151-152 and 171-173), suggesting that too many consecutive 2′-O-methylmodifications at the 5′ end (e.g., 26 or more consecutive2′-O-methyl-modified nucleotides), or too many 2′-O-methyl modificationsof C and U nucleotides downstream (3′) of the 5′-terminal 20 mer guidesequence is not well tolerated (e.g., the inhibitory effect of one orboth 2′-O-methyluracils at sequence positions +56 and +69 in entries171-173 as revealed by the positions tested in entries 154-156).

The present example shows that 2-O-methyl modifications throughout the20 mer guide sequence are tolerated during in vitro uses in buffercontaining 10 mM Mg2+ (entry 146), but such extensive modification isnot well tolerated under physiological conditions (entries 147-150) aspresent in cells. Thus, in some embodiments, a gRNA comprising 15 ormore, alternatively 17 or more, alternatively 18 or more, alternatively20 2′-O-methyl modifications throughout the 20 mer guide sequence isused for in vitro methods as described herein, such as genomic editingto modify a DNA sequence in vitro, regulating the expression of a geneof interest in vitro, cleaving a DNA target sequence in vitro, and otheruses.

The present example shows that extensive incorporation of 2′-deoxymodifications is not well tolerated and can be substantially completelyinhibitory (entries 180-182). However, 2′-deoxy modifications can bewell-tolerated at some locations (entry 183), therefore suchmodification can be useful for inhibiting nucleases.

The present example also shows that fluorophore or dye labels aretolerated in every loop of the three known stem-loops in CRISPR-Cas9guide RNAs (entry 116). Such labels are also tolerated in a 5′ overhangon the guide sequence (entry 114), tolerated at additional locations insgRNAs (entry 114), and tolerated in a loop in tracrRNA used indual-guide applications (entry 11). In this example, two different typesof fluorophore were tested: a phosphodiester-linked fluorophore (noribose ring) that essentially takes the place of a nucleotide (entries114 & 116), and a dye label (Cy5) covalently coupled to 5-aminoallylUincorporated in a guide RNA (entry 11).

The present example also shows that Z bases are tolerated in syntheticguide RNAs, particularly as modifications of synthetic guide RNAs inwhich some C's are replaced with Z bases (entries 290-291). The presentexample also shows that several other bases are tolerated at variouspositions, as shown in Tables 3 and 4.

The present example further shows that the 5′ and 3′ ends of guide RNAscan tolerate a wide variety of end-protecting modifications. Suchmodifications can be used to inhibit exonucleolytic RNA degradation.Support for the tolerance of such modifications can be found in Hendelet al., Nat. Biotechnol. (2015) 55:9, 985-9. Additional support formodifications at the 5′ and 3′ ends of guide RNAs is provided by entries143-144, 185-223, 241-257, 258-266, and 272-279 in Tables 3 and 4. Insome embodiments, the guide RNA comprises 7 or fewer modifiednucleotides at a 5′ end or a 3′ end or at each of 5′ and 3′ ends,alternatively 6 or fewer, alternatively 5 or fewer, alternatively 4 orfewer, alternatively 3 or fewer, alternatively 2 or fewer,alternatively 1. dual-guide RNAs can be protected similarly (entries12-15).

The present example further shows that 2-thioU can be used to deteroff-target interactions involving G-U wobble pairings, therebyincreasing the specificity of guide sequence pairing by inhibitingoff-target hybridization interactions (entries 16-39). One of the basepairs involved in hybridization between the guide RNA and CLTA1OFF-target 3 (also referred to as “CLTA1 OFF3-target” or “CLTA1 OFF3”)is a G-U wobble pair. Replacing the corresponding U in the guide RNAwith a 2-thioU reduces cleavage from 100% (entry 8) to 59% (entry 39).Replacing other U's with 2-thioU's (e.g., at sequence position +3 or +9,entries 23 and 31) does not have the same effect, presumably becausethose U's do not involve G-U wobble pairing when fully hybridized toeach of the OFF-target sites tested. Accordingly, 2-thioU can increasetarget specificity of guide RNAs when off-target sites involve G-Uwobble pairing.

The present example also shows that 5′-overhang sequences attached tothe guide sequence are generally well-tolerated (see entries 83-95, 114,and 206-223). For example, a bulky dimethoxytrityl (dmt) group at the 5′end was well tolerated (entry 95). The chromatographic properties of dmtcan be used to facilitate purification of full-length synthetic RNAsfrom incompletely elongated byproducts which are generally producedduring synthesis. Accordingly, in some embodiments, the synthetic guideRNA comprises a 5-overhang sequence, for example, comprising a shortpolynucleotide sequence of 15 or fewer nucleotides which iscomplementary to the guide sequence and is covalently linked at its 3′end to the 5′ end of the guide sequence by a polymeric linker such as apolynucleotide or similarphosphodiester-based linker, in which thelinker can be 5 or more consecutive uridine nucleotides, alternatively 6or 7.

The present example also shows that surface exposed regions of the guideRNA (as inferred from crystal structures published by others) aretolerant of extensively modifying uracils nucleotides to 5-methyluracils(5-methylU's) (entry 288), which can make the modified RNA more likelyto elude immune responses such as stimulated by unmodified RNA. Inparticular, the 5′ and 3′ ends of a synthetic guide RNA are potentiallyimmunostimulatory, and the present example shows that 5′ and 3′ ends aretolerant of 5-methylU modifications (entry 288).

The present example also shows that a synthetic guide RNA is tolerant ofreplacing some G-C pairs with 2-O-methylA-2′-O-methylU pairs which aremore stable thermodynamically than unmodified A-U pairs (sec thenon-terminal-2′-O-methylU and complementary -2′-O-methylA modificationsin entries 292-293). This is advantageous because it is known that, forfolded RNAs, G-C pairs are stronger and more stable than A-U pairs.Replacement of G-C pairs with such thermostabilized A-U pairs insynthetic guide RNAs allows for improved folding of active structures bypreventing misfolded structures that involve unintended G-C pair(s), ascan be predicted by RNA folding algorithms in common use.

Exemplary Embodiments

Exemplary embodiments provided in accordance with the presentlydisclosed subject matter include, but are not limited to, the claims andthe following embodiments:

A1. A synthetic guide RNA comprising:

-   -   a crRNA segment comprising (i) a guide sequence capable of        hybridizing to a target sequence, (ii) a stem sequence; and    -   a tracrRNA segment comprising a nucleotide sequence that is        partially or completely complementary to the stem sequence,        wherein the synthetic guide RNA comprises at least one modified        nucleotide, and wherein the synthetic guide RNA has gRNA        functionality.

A2. The synthetic guide RNA of embodiment A1, comprising a 2-deoxymoiety.

A3. The synthetic guide RNA of embodiment A1 or A2, comprising a 2-halomoiety selected from 2′-fluoro, 2′-chloro, 2′-bromo and 2′-iodo.

A4. The synthetic guide RNA of any one of the preceding embodiments,comprising a phosphorothioate group.

A5. The synthetic guide RNA of any one of the preceding embodiments,comprising a PACE group.

A6. The synthetic guide RNA of any one of the preceding embodiments,comprising a thioPACE group;

A7. The synthetic guide RNA of any one of embodiments A2-A6, comprisinga 2′-O-methyl moiety.

A8. The synthetic guide RNA of any one of the preceding embodiments,comprising 2-thiouracil.

A9. The synthetic guide RNA of any one of the preceding embodiments,comprising,a 4-thiouracil.

A10. The synthetic guide RNA of any one of the preceding embodiments,comprising a 2-aminoadenine.

A11. The synthetic guide RNA of any one of the preceding embodiments,comprising a hypoxanthine.

A12. The synthetic guide RNA of any one of the preceding embodiments,comprising a 5-methylcytosine.

A13. The synthetic guide RNA of any one of the preceding embodiments,comprising a 5-methyluracil.

A14. The synthetic guide RNA of any one of the preceding embodiments,comprising a 5-aminoallyl-uracil.

A15. The synthetic guide RNA of any one of the preceding embodiments,comprising a Z ribonucleotide.

A16: The synthetic guide RNA of any one of the preceding embodiments,comprising a Z deoxyribonucleotide.

A17. The synthetic guide RNA of any one of the preceding embodiments,comprising a squarate conjugation.

A18. The synthetic guide RNA of any one of the preceding embodiments,comprising a dye linker.

A19. The synthetic guide RNA of embodiment A18, wherein the dye linkeris 2-(4-butylamidofluorescein)propane-1,3-diol bis(phosphodiester)linker.

A20: The synthetic guide RNA of any one of the preceding embodiments,comprising a nucleotide with 2′-O-methyl and 3′-phosphorothioate.

A21. The synthetic guide RNA of any one of the preceding embodiments,comprising a nucleotide with 2′-O-methyl and 3′-PACE.

A22. The synthetic guide RNA of any one of the preceding embodiments,comprising a nucleotide with 2-O-methyl and 3-thioPACE.

A23. The synthetic guide RNA of any one of the preceding embodiments,comprising a nucleotide with 2′-deoxy and 3-PACE.

A24. The synthetic guide RNA of any one of the preceding embodiments,comprising a 5-methylcytidine.

A25. The synthetic guide RNA of any one of the preceding embodiments,comprising a methylphosphonate.

A26. The synthetic guide RNA of any one of the preceding embodiments,comprising an ester of PACE, wherein the ester is optionally a methylester.

A27. The synthetic guide RNA of any one of the preceding embodiments,comprising a single RNA strand comprising both the crRNA segment and thetracrRNA segment.

A28. The synthetic guide RNA of any one of embodiments A1-A26,comprising two RNA strands, and the crRNA segment and the tracrRNAsegment are in different RNA strands.

A29. The synthetic guide RNA of any one of the preceding embodiments,comprising a modified nucleotide at a 5′ end, 3′ end, or both 5′ end and3′ end of each RNA strand.

A30. The synthetic guide RNA of any one of the preceding embodiments,comprising a modified nucleotide in the guide sequence.

A31. The synthetic guide RNA of any one of the preceding embodiments,comprising a modified nucleotide 5′ to the guide sequence.

A32. The synthetic guide RNA of any one of the preceding embodiments,comprising a modified nucleotide in the stem sequence.

A33. The synthetic guide RNA of any one of the preceding embodiments,comprising a modified nucleotide in the scaffold region.

A34. The synthetic guide RNA of any one of the preceding embodiments,comprising at least one unnatural, orthogonal base pair in the scaffoldregion, wherein the base pair is independently selected from isoG-isoCand Z base-P base.

A35. The synthetic guide RNA of any one of the preceding embodiments,comprising a 2′-amino group.

A36. The synthetic guide RNA of any one of the preceding embodiments,comprising a phosphorothioate linkage group.

A37. The synthetic guide RNA of any one of the preceding embodiments,comprising a boranophosphonate linkage group.

A38. The synthetic guide RNA of any one of the preceding embodiments,comprising an unlocked nucleic acid modification (ULNA).

A39. The synthetic guide RNA of any one of the preceding embodiments,comprising a locked nucleic acid modification (LNA).

A40. The synthetic guide RNA of any one of the preceding embodiments,comprising an unstructured nucleic acid modification (UNA).

A41. The synthetic guide RNA of any one of the preceding embodiments,comprising a pseudoU.

A42. The synthetic guide RNA of any one of the preceding embodiments,comprising a 2-MOE.

A43. The synthetic guide RNA of any one of the preceding embodiments,comprising a 2′-arabino.

A44. The synthetic guide RNA of any one of the preceding embodiments,comprising a 4′-thioribose.

A45. The synthetic guide RNA of any one of the preceding embodiments,comprising a squarate linkage

A46. The synthetic guide RNA of any one of the preceding embodiments,comprising a triazaolo linkage.

A47. A method for cleaving or nicking a target polynucleotide comprisingcontacting the target polynucleotide with a CRISPR-associated proteinand the synthetic guide RNA of any one of the preceding embodiments, andcleaving or nicking the target polynucleotide.

A48. The method of embodiment A47, wherein the cleaving or nicking takesplace in vitro.

A49. The method of embodiment A47, wherein the cleaving or nicking takesplace in a cell.

A50. The method of embodiment A47, wherein the cleaving or nicking takesplace in vivo.

A51. The method of any one of embodiments A47-A50, wherein theCRISPR-associated protein is Cas9.

A52. The method of any one of embodiments A47-A51, wherein the cleavingor nicking results in gene editing.

A53. The method of any one of embodiments A47-A52, wherein the cleavingor nicking results in alteration of gene expression.

A54. A method for binding a target polynucleotide comprising contactingthe target polynucleotide with a CRISPR-associated protein and thesynthetic guide RNA of any one of the preceding embodiments.

A55. The method of embodiment A54, wherein the CRISPR-associated proteincomprises a mutant which does not have a cleavage or nicking activity.

A56. The method of embodiment A54 or A55, wherein the CRISPR-associatedprotein is a fusion protein comprising a protein component not naturallyexisting in a CRISPR system.

A57. The method of any one of embodiments A54 to A56, resulting in achange of expression of the target polynucleotide.

A58. The method of any one of embodiments A54 to A57 useful to tag thetarget polynucleotide.

Further Exemplary Embodiments

B1. A synthetic guide RNA comprising:

-   -   (a) a crRNA segment comprising (i) a guide sequence capable of        hybridizing to a target sequence in a polynucleotide, (ii) a        stem sequence; and    -   (b) a tracrRNA segment comprising a nucleotide sequence that is        partially or completely complementary to the stem sequence,        wherein the synthetic guide RNA comprises one or more        modifications, and wherein the synthetic guide RNA has-gRNA        functionality.

B2. The synthetic guide RNA of embodiment 1, comprising a 2′-O-methylmoiety, a 2′-deoxy moiety, a Z base, a phosphorothioate internucleotidelinkage, a phosphonoacetate internucleotide linkage, athiophosphonoacetate internucleotide linkage, or combinations thereof.

B3. The synthetic guide RNA of embodiment 1 or 2, comprising one or moremodifications selected from the group consisting of a 2′-O-methylnucleotide with a 3′-phosphorothioate group, a 2-O-methyl nucleotidewith a 3′-phosphonocarboxylate group, a 2′-O-methyl nucleotide with a3′-phosphonoacetate group, a 2′-O-methyl nucleotide with a3′-thiophosphonocarboxylate group, a 2′-O-methyl nucleotide with a3′-thiophosphonoacetate group, a 2′-deoxy nucleotide with a3′-phosphonoacetate group, a 2′-deoxy nucleotide with a3′-thiophosphonoacetate group, and a Z base.

B4. The synthetic guide RNA of embodiment 1, 2 or 3, comprising one ormore modifications selected from the group consisting of a2′-fluororibosyl, a 2-thiouracil base, a 4-thiouracil base, a2-aminoadenine base, an hypoxanthine base, a 5-methylcytosine base, a5-methyluracil base, a methylphosphonate internucleotide linkage, a5-aminoallyluracil base, a squarate linkage, a triazolo linkage, a dyeconjugated to a nucleotide, and combinations thereof.

B5. The synthetic guide RNA of any of the preceding embodiments,comprising a modification selected from the group consisting of a2′-MOE, 2′-amino, 2′-F-arabino, 2′-LNA, 2′-ULNA, 3′-methylphosphonate,3′-boranophosphonate, 3′-phosphorodithipate, 2′-OMe-3′-P(S)₂,2′-OMe-3′-P(CH₃), 2′-OMe-3′-P(BH₃), 4-thioribosyl, L-sugar,2-thiocytosine, 6-thioguanine, 2-aminopurine, pseudouracil,7-deazaguanine, 7-deaza-8-azaguanine, 7-deazaadenine,7-deaza-8-azaadenine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil,5,6-dehydrouracil, 5-propynylcytosine, 5-propynyluracil,5-ethynylcytosine, 5-ethynyluracil, 5-allyluracil, 5-allylcytosine,5-allylaminocytosine, P nucleobase, isocytosine (isoC), isoguanine(isoG), UNA, x(A,G,C,T), y(A,G,C,T), abasic nucleotide, PEG, hydrocarbonlinker, halo-substituted hydrocarbon linker, heteroatom(O,N,S)-substituted; hydrocarbon linker, (keto, carboxy, amido, thionyl,carbamoyl, or thionocarbamoyl)-containing hydrocarbon linker, sperminelinker, and combinations thereof.

B6. The synthetic guide RNA of any one of the preceding embodiments,comprising a stability-enhancing modification.

B7. The synthetic guide RNA of any one of the preceding embodiments,comprising at least two modifications; wherein a first modification is astability-enhancing modification and a second modification is aspecificity-altering modification.

B8. The synthetic guide RNA of embodiment 6 or 7, wherein thestability-enhancing modification is located in the guide sequence.

B9. The synthetic guide RNA of embodiment 6 or 7, wherein thestability-enhancing modification is located upstream of the guidesequence.

B10. The synthetic guide RNA of embodiment 6 or 7, wherein thestability-enhancing modification is located within the first five and/orthe last five nucleotides of the crRNA segment.

B11. The synthetic guide RNA of embodiment 6 or 7, wherein thestability-enhancing modification is located in the tracrRNA segment.

B12. The synthetic guide RNA of embodiment 6 or 7, wherein thestability-enhancing modification is located within the first five and/orthe last five nucleotides of the tracrRNA segment.

B13. The synthetic guide RNA of any one of embodiments 6-12, wherein thestability-enhancing modification comprises a 2′-O-methyl moiety, a2′-fluoro moiety, or a 2-deoxy moiety.

B14. The synthetic guide RNA of any one of embodiments 6-13, wherein thestability-enhancing modification comprises a phosphorothioateinternucleotide linkage, a phosphonoacetate internucleotide linkage, athiophosphonoacetate internucleotide linkage, a methylphosphonateinternucleotide linkage, a boranophosphate internucleotide linkage, or aphosphorothioate internucleotide linkage.

B15. The synthetic guide RNA of any one of embodiments 6-14, wherein thestability-enhancing modification comprises a 3′-phosphonoacetate or a3′-thiophosphonoacetate.

B16. The synthetic guide RNA any one of embodiments 6-15, wherein thestability-enhancing modification comprises a2-O-methyl-3′-phosphorothioatenucleotide, a2′-O-methyl-3′-phosphonoacetate nucleotide, or a2′-O-methyl-3′-thiophosphonoacetate nucleotide.

B17. The synthetic guide RNA of any one of embodiments 6-16, wherein thestability-enhancing modification comprises a2′-fluoro-3′-phosphorothioate nucleotide, a2′-fluoro-3′-phosphonoacetate nucleotide, or a2′-fluoro-3′-thiophosphonoacetate nucleotide.

B18. The synthetic guide RNA of any one of the preceding embodiments,comprising a specificity-altering modification.

B19. The synthetic guide RNA of embodiment 18, wherein thespecificity-altering modification is located in the guide sequence.

B20. The synthetic guide RNA of any one of embodiment 18 or 19, whereinthe specificity-altering modification comprises a 2-thiouracil, a4-thiouracil or a 2-aminoadenine.

B21. The synthetic guide RNA of any one of embodiments 18-20, whereinthe specificity-altering modification comprises a phosphorothioateinternucleotide linkage, a phosphonoacetate internucleotide linkage, athiophosphonoacetate internucleotide linkage, a methylphosphonateinternucleotide linkage, a boranophosphate internucleotide linkage, or aphosphorothioate internucleotide linkage.

B22. The synthetic guide RNA of any one of embodiments 18-21, whereinthe specificity-altering modification comprises a 3′-phosphonoacetate ora 3-thiophosphonoacetate.

B23. The synthetic guide RNA of any one of the preceding embodiments,comprising a fluorescent dye or a label.

B24. The synthetic guide RNA of any one of the preceding embodiments,comprising one or more isotopic labels.

B25. The synthetic guide RNA of any one of the preceding embodiments,wherein the guide RNA is conjugated to an oligonucleotide, an aptamer,an amino acid; a peptide, a protein, a steroid, a lipid, a folic acid, avitamin, a sugar, or an oligosaccharide.

B26. The synthetic guide RNA of any one of the preceding embodiments,wherein the synthetic guide RNA is a single guide RNA, wherein the crRNAsegment and the tracrRNA segment are linked through a loop L.

B27. The synthetic guide RNA of embodiment 26, wherein the loop Lcomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.

B28. The synthetic guide RNA of embodiment 26 or 27, wherein the loop Lcomprises a nucleotide sequence of GNRA, wherein N represents A, C, G,or U and R represents A or G.

B29. The synthetic guide RNA of embodiment 26, 27 or 28, wherein theloop L comprises a nucleotide sequence of GAAA.

B30. The synthetic guide RNA of any one of embodiments 26-29, whereinthe loop L comprises one or more modified nucleotides.

B31. The synthetic guide RNA of embodiment 30, wherein the loop Lcomprises a fluorescent dye.

B32. The synthetic guide RNA of embodiment 31, wherein the dye isconjugated to a 2-(4-butylamido-dye)propane-1,3-diol bis(phosphodiester)linker.

B33. The synthetic guide RNA of any one of the preceding embodiments,wherein the crRNA segment is at the 5′ end of the guide RNA.

B34. The synthetic guide RNA of any one of the preceding embodiments,wherein the tracrRNA segment is at the 3′ end of the guide RNA.

B35. The synthetic guide RNA of any of the preceding embodiments,wherein the crRNA segment comprises from 25 to 70 nucleotides.

B36. The synthetic guide RNA of any of the preceding embodiments,wherein the guide sequence comprises from 15 to 30 nucleotides.

B37. The synthetic guide RNA of any of the preceding embodiments,wherein the stem sequence comprises from 10 to 50 nucleotides.

B38. The synthetic guide RNA of any of the preceding embodiments,comprising one or more triazolo linkage(s).

B39. The synthetic guide RNA of any of the preceding embodiments,comprising one or more squarate linkage(s).

B40. The synthetic guide RNA of any of the preceding embodiments,wherein the guide RNA comprises a nucleotide composition of:

MmNn

wherein each N independently represents an unmodified nucleotide andeach M is selected from a 2′-O-methyl ribonucleotide, a2′-O-methyl-3′-P(S) ribonucleotide, a 2′-O-methyl-3′-PACEribonucleotide, a 2′-O-methyl-3′-thioPACE ribonucleotide, and a2′-deoxynucleotide;

wherein each M is at any position in the sequence of the guide RNA; and

wherein m is an integer between 1 and 220, and n is an integer between 0and 219, and 50<m+n≦220.

B41. The synthetic guide RNA of embodiment 38, wherein m+n <150, andeach of m and n are independently selected from an integer between 0 and150, provided that m is not 0.

B42. The synthetic guide RNA of any of the preceding embodiments,wherein the guide RNA comprises a nucleotide sequence of:

M_(m)N_(n)M′_(m′)N′_(n′)M″_(m″)

wherein each M, M′ and M″ independently represent a modified nucleotideand each N and N′ independently represent an unmodified ribonucleotide;

wherein any given M is the same or different from any other M, any givenN is the same or different from any other N, any given M′ is the same ordifferent from any other M′, any given N′ is the same or different fromany other N′, any given M″ is the same or different from any other M″;and

wherein m is an integer between 0 and 40, n is an integer between 20 and130, m′ is an integer between 0 and 10, n′ is an integer between 0 and50, m″ is an integer between 0 and 10, provided that m+m′+m″ is greaterthan or equal to 1.

B43. The synthetic guide RNA of any of the preceding embodiments,wherein the crRNA segment comprises a nucleotide sequence of:

M_(m)N_(n)M′_(m′)N′_(n′)

wherein M and M′ each represent a modified nucleotide and N and N′ eachrepresent an unmodified ribonucleotide;

wherein any given M is the same or different from any other M, any,given N is the same or different from any other N, any given M′ is thesame or different from any other M′, any given N′ is the same ordifferent from any other N′; and

wherein n and n′ are each independently selected from an integer between0 and 50, and wherein m and m′ are each independently selected from aninteger between 0 and 25, provided that m+m′ is greater than or equal to1.

B44. The synthetic guide RNA of any of the preceding embodiments,wherein the guide sequence comprises a nucleotide sequence of:

M_(m)N_(n)M′_(m′)N′_(n′)

wherein M and M′ each represent a modified nucleotide and N and N′ eachrepresent an unmodified ribonucleotide;

wherein any given M is the same or different from any other M, any givenN is the same or different from any other N, any given M′ is the same ordifferent from any other M′, any given N′ is the same or different fromany other N′; and

wherein m, n, m′, and n′ are each independently selected from an integerbetween 0 and 40, provided that m+m′ is greater than or equal to 1.

B45. The synthetic guide RNA of any of the preceding embodiments,wherein the tracrRNA segment comprises a nucleotide sequence of:

N_(n)M_(m)N′_(n′)M′_(m′)

wherein M and M′ each represent a modified nucleotide and N and N′ eachrepresent an unmodified ribonucleotide;

wherein any given M is the same or different from any other M, any givenN is the same or different from any other N, any given M′ is the same ordifferent from any other M′, any given N′ is the same or different fromany other N′; and

wherein n is an integer between 0 and 130 m is an integer between 0 and40, and n′ is an integer between 0 and 130, and m′ is an integer between0 and 40, provided that m+m′ is greater than or equal to 1.

B46. The synthetic guide RNA of any one of embodiments 40-43, wherein m,m′, m+m′, or m+m′+m″ is 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20.

B47. The synthetic guide RNA of any one of embodiments 40-43, wherein m,m′, m+m′, or m+m′+m″ is 1, 2, 3, 4, 5, or 6.

B48. The synthetic guide RNA of any one of embodiments 40-45, wherein nis 16, 17, 18, or 19.

B49. The synthetic guide RNA of any one of embodiments 40-45, wherein n,n′, or n+n′ is an integer between 75 and 115.

B50. The synthetic guide RNA of any one of embodiments 40-47, whereineach M is independently selected from the group consisting of a2′-modified nucleotide, a 3′-modified nucleotide, and combinationsthereof.

B51. The synthetic guide RNA of embodiment 48, wherein the 2′-modifiednucleotide is selected from the group consisting of a 2′-deoxynucleotide, a 2′-O-methyl nucleotide, a 2′-fluoro nucleotide, and a2′-O-C₁₋₃alkyl-O-C₁₋₃alkyl nucleotide.

B52. The synthetic guide RNA of embodiment 48, wherein the 3′-modifiednucleotide is selected from the group consisting of a3′-phosphonoacetate nucleotide and a 3′-thiophosphonoacetate nucleotide.

B53. The synthetic guide RNA of embodiment 48, wherein the combinationof the 2′-modified nucleotide and the 3′-modified nucleotide comprises a2′-O-methyl-3′-phosphorothioate nucleotide, a2′-O-methyl-3-phosphonoacetate nucleotide, or a2′-O-methyl-3′-thiophosphonoacetate nucleotide.

B54. A method for cleaving a target polynucleotide comprising contactingthe target polynucleotide with a CRISPR-associated protein and thesynthetic guide RNA of any one of the preceding embodiments and cleavingthe target polynucleotide.

B55. The method of embodiment 52, further comprising contacting thetarget polynucleotide with an exogenous CRISPR-associated protein.

B56. The method of embodiment 53, wherein the CRISPR-associated proteinis Cas9.

B57. The method of any one of embodiments 52-54, wherein the cleavageresults in a functional knockout of a target gene.

B58. The method of any one of embodiments 52-55, further comprisingrepairing the cleaved target polynucleotide by homology-directed repairwith an exogenous or endogenous template polynucleotide.

B59. The method of embodiment 56, wherein the exogenous or endogenoustemplate polynucleotide comprises at least one sequence havingsubstantial sequence identity with a sequence on either side of thecleavage site.

B60. The method of any one of embodiments 52-57, further comprisingrepairing the cleaved target polynucleotide by non-homologous endjoining.

B61. The method of any one of embodiments 56-58, wherein the repairingstep produces an insertion, deletion, or substitution of one or morenucleotides of the target polynucleotide.

B62. The method of embodiment 59, wherein the insertion, deletion, orsubstitution results in one or more amino acid changes in a proteinexpressed from a gene comprising the target polynucleotide.

B63. The method of any one of embodiments 52-60, wherein the targetpolynucleotide is contacted with the CRISPR-associated protein and thesynthetic guide RNA in vitro.

B64. The method of any one of embodiments 52-61, wherein the targetpolynucleotide contacted with the CRISPR-associated protein and thesynthetic guide RNA is within the genome of a cell in vitro or in vivo.

B65. The method of embodiment 62, wherein the cell is isolated from amulticellular source prior to contacting the target polynucleotide withthe CRISPR-associated protein and the synthetic guide RNA.

B66. The method of embodiment 63, wherein the source is a plant, ananimal, a multicellular protist, or a fungus.

B67. The method of any one of embodiments 62-64, wherein the cell, or acell derived therefrom, is returned to the source after contacting thetarget polynucleotide with the CRISPR-associated protein and thesynthetic guide RNA.

B68. A method of modifying a target polynucleotide in a cell comprisingintroducing into the cell the synthetic guide RNA of any one ofembodiments 1-51 and introducing into the cell a CRISPR-associatedprotein or a nucleic acid that expresses a CRISPR-associated protein inthe cell.

B69. The method of embodiment 66, wherein the CRISPR-associated-proteinis Cas9.

B70. A method of altering expression of at least one gene product in acell comprising introducing into the cell the synthetic guide RNA of anyone of embodiments 1-51 and further introducing into the cell aCRISPR-associated-protein or a nucleic acid that expresses aCRISPR-associated protein in the cell, wherein the cell contains andexpresses a DNA molecule having a target sequence and encoding the geneproduct.

B71. The method of embodiment 68, wherein the CRISPR-associated-proteinis Cas9.

B72. The method of embodiment 69, wherein the CRISPR-associated-proteincleaves the DNA molecule.

B73. A set or library of RNA molecules comprising two or moresynthetic,guide RNAs of any one of embodiments 1-51.

B74. A kit comprising the synthetic guide RNA of any one of embodiments1-51 or the set or library of RNA molecules of embodiment 71.

B75. The kit of embodiment 72, further comprising a CRISPR-associatedprotein or a nucleic acid encoding the CRISPR-associated protein.

B76. The kit of embodiment 73, wherein the CRISPR-associated-protein isCas9.

B77. The synthetic guide RNA, method or kit of any of the precedingembodiments, wherein the synthetic guide RNA comprises an endmodification.

B78. The synthetic guide RNA of any of the preceding embodiments, havinga single RNA strand or two separate complementary RNA strands, whereinthe guide RNA comprises at least one stability-enhancing modification atboth ends of each RNA strand;

C1. A synthetic guide RNA comprising:

-   -   (a) a crRNA segment comprising (i) a guide sequence capable of        hybridizing to a target sequence in a polynucleotide, (ii) a        stem sequence; and    -   (b) a tracrRNA segment comprising a nucleotide sequence that is        partially or completely complementary to the stem sequence,        wherein the synthetic guide RNA comprises one or more        modifications, and wherein the synthetic guide RNA has gRNA        functionality.

C2. The synthetic guide RNA of embodiment C1, wherein one or more of themodifications comprises a stability-enhancing modification.

C3. The synthetic guide RNA of embodiment C2, wherein one-or more of thestability-enhancing modifications is located in the guide sequence.

C4. The synthetic guide RNA of embodiment C2, wherein thestability-enhancing modification comprises a 2′-O-methyl moiety, a Zbase, a 2′-deoxy nucleotide, a phosphorothioate internucleotide linkage,a phosphonoacetate (PACE) internucleotide linkage, or athiophosphonoacetate (thioPACE) internucleotide linkage, or combinationsthereof.

C5. The synthetic guide RNA of any of the foregoing embodiments,comprising less than 26 consecutive 2′-O-methyl modified nucleotides ata 5′ end of the guide RNA.

C6. The synthetic guide RNA of any of the foregoing embodiments,comprising a Z base replacing a cytosine in the synthetic guide RNA.

C7. The synthetic guide RNA of any of the foregoing embodiments,comprising at least one 2-thiouracil at a position corresponding to auridine that can engage in U-G wobble pairing with a potentialoff-target sequence.

C8. The synthetic guide RNA of any of the foregoing embodiments,comprising one or more modifications selected from the group consistingof a 2′-O-methyl nucleotide with a 3′-phosphorothioate group, a2′-O-methyl nucleotide with a 3′-phosphonoacetate group, a 2′-O-methylnucleotide with a 3′-thiophosphonoacetate group, and a 2′-deoxynucleotide with a 3′-phosphonoacetate group.

C9. The synthetic guide RNA of any of the foregoing embodiments,comprising at least two modifications.

C10. The synthetic guide RNA any of the foregoing embodiments,comprising up to 50 modifications.

C11. The synthetic guide RNA of any of the foregoing embodiments,comprising a single RNA strand or two separate RNA strands, and one ormore modifications at a 5′ end of each RNA strand, at a 3′ end of eachRNA strand, or at both a 5′ end and a 3′ end of each RNA strand.

C12. The synthetic guide RNA of any of the foregoing embodiments,comprising 7 or fewer consecutive modified nucleotides at a 5′ end or ata 3′ end or at each of 5′ and 3′ ends.

C13. The synthetic guide RNA of any of the foregoing embodiments,comprising one or more 5-methyluridine nucleotides at one or more of a5′ end, a 3′ end or a stem-loop.

C14. The synthetic guide RNA of any of the foregoing embodiments,wherein one or more of the modifications alters base-pairingthermostability.

C15. The synthetic guide RNA of embodiment C14, wherein said one or moremodifications enhances the base-pairing thermostability.

C16. The synthetic guide RNA of embodiment C15, wherein said one or moremodifications is independently selected from a 2-thiouracil (2-thioU), a4-thiouracil (4-thioU), a 2-aminoadenine, a 2′-O-methyl, a 2′-fluoro, a5-methyluridine, a 5-methylcytidine, and a locked nucleic acidmodification (LNA).

C17. The synthetic guide RNA of embodiment C15, wherein said one or moremodifications decreases the base-pairing thermostability.

C18. The synthetic guide RNA of embodiment C17, wherein said one or moremodifications is independently selected from a 2-thiouracil, a 2′-deoxy,a phosphorothioate linkage, a phosphorothioate linkage, aboranophosphonate linkage, a phosphonoacetate linkage, athiophosphonoacetate linkage, and an unlocked nucleic acid modification(ULNA).

C19. The synthetic guide RNA of any of the foregoing embodiments,comprising one or more 2′-O-methylA-2′-O-methylU base pairs.

C20. The synthetic guide RNA of any of the foregoing embodiments,wherein one or more of the modifications is a specificity-alteringmodification.

C21. The synthetic guide RNA of embodiment C20, wherein thespecificity-altering modification is located in the guide sequence.

C22. The synthetic guide RNA of any of the foregoing embodiments,wherein the specificity-altering modification comprises a 2-thiouracil,a 4-thiouracil, a 2-aminoadenine, a 2′-O-methyl, a 2′-fluoro, a LNA, aphosphorothioate linkage, a phosphorothioate linkage, aboranophosphonate linkage, a phosphonoacetate linkage, athiophosphonoacetate linkage, an ULNA, a 2′-deoxy, a 5-methyluridine, a5-methylcytidine, or combinations thereof.

C23. The synthetic guide RNA of any of the foregoing embodiments,comprising a fluorescent dye or a label.

C24. The synthetic guide RNA of embodiment C23, wherein the fluorescentdye or a label is bound to a stem-loop of the synthetic guide RNA.

C25. A method for genomic editing to modify a DNA sequence, orregulating the expression of a gene of interest, or cleaving a targetpolynucleotide comprising: contacting the DNA sequence, the gene ofinterest, or the target polynucleotide with a CRISPR-associated proteinand the synthetic guide RNA of any of the foregoing embodiments, andediting, regulating or cleaving the DNA sequence, the gene of interestor the target polynucleotide.

C26. The method of embodiment C25, wherein the method is performed invitro, and the synthetic guide RNA comprises 15 or more 2′-O-methylmodifications throughout the guide sequence.

C27. A set or library of RNA molecules comprising two or more syntheticguide RNAs of any of the foregoing embodiments.

C28. A kit comprising the synthetic guide RNA of any of the foregoingembodiments.

C29. An array of RNA molecules comprising two or more, synthetic, guideRNAs of any of the foregoing embodiments.

The foregoing description of exemplary or preferred embodiments shouldbe taken as illustrating, rather than, as limiting, the presentinvention as defined by the claims. As will be readily appreciated,numerous variations and combinations of the features set forth above canbe utilized without departing from the present invention as set forth inthe claims. Such variations are not regarded as a departure from thescope of the invention, and all such variations are intended to beincluded within the scope of the following claims. All references citedherein are incorporated by reference in their entireties.

We claim:
 1. A synthetic guide RNA having at least one 5′-end and atleast one 3′-end, the synthetic guide RNA comprising: (a) one or moremodified nucleotides within five nucleotides from said 5′-end, or (b)one or more modified nucleotides within five nucleotides from said3′-end, or (c) both (a) and (b); wherein said guide RNA comprises one ormore RNA molecules and has gRNA functionality.
 2. The synthetic guideRNA of claim 1 wherein said guide RNA is a single guide RNA (sgRNA). 3.The synthetic guide RNA of claim 1 wherein said guide RNA comprises: (a)one modified nucleotide at the first nucleotide of said 5′-end, or (b)at least one modified nucleotide within two nucleotides from said3′-end, or (c) both (a) and (b).
 4. The synthetic guide RNA of claim 1wherein said modified nucleotide comprises a modified internucleotidelinkage or a modified terminal phosphate group selected from analkylphosphonate, a phosphonocarboxylate, a phosphonoacetate, aboranophosphonate, a phosphorothioate, a phosphonothioacetate, and aphosphorothioate group.
 5. The synthetic guide RNA of claim 1 whereinsaid guide RNA comprises: (a) one or more modified nucleotides withinfive nucleotides from said 5′-end, or (b) at least two consecutivemodified nucleotides within five nucleotides from said 3′-end, or (c)both (a) and (b).
 6. The synthetic guide RNA of claim 1 wherein saidguide RNA comprises a modified internucleotide linkage or a modifiedterminal phosphate group selected from a phosphonocarboxylate, aphosphonoacetate, and a phosphonothioacetate group.
 7. The syntheticguide RNA of claim 1 wherein said one or more modified nucleotidescomprise a 2′-O-methyl-3′-phosphorothioate nucleotide.
 8. The syntheticguide RNA of claim 1 wherein said one or more modifications areindependently selected from a 2′-deoxy, 2′-O-methyl,3′-phosphorothioate, 3′-phosphonoacetate (PACE), 3′-phosphonothioacetate(thioPACE), and a combination thereof.
 9. The synthetic guide RNA ofclaim 8 wherein said one or more modified nucleotides comprise a2′-O-methyl-3′-phosphonoacetate nucleotide or a2′-O-methyl-3′-phosphonothioacetate nucleotide.
 10. The synthetic guideRNA of claim 3 wherein said one modified nucleotide is independentlyselected from a 2′-O-methyl nucleotide, 2′-O-methyl-3′-phosphorothioatenucleotide, 2′-O-methyl-3′-phosphonoacetate nucleotide, and2′-O-methyl-3′phosphonothioacetate nucleotide.
 11. The synthetic guideRNA of claim 5 wherein said one or more modified nucleotides within fivenucleotides from the 3′-end are each independently selected from a2′-O-methyl nucleotide, 2′-O-methyl-3′-phosphorothioate nucleotide,2′-O-methyl-3′-phosphonoacetate nucleotide,2′-O-methyl-3′-phosphonothioacetate nucleotide, and a combinationthereof.
 12. A synthetic crRNA molecule comprising a 5′-end, a3′-end,and a guide sequence capable of hybridizing to a targetpolynucleotide, wherein said crRNA molecule comprises: (a) one or moremodified nucleotides within five nucleotides from said 5′-end, or (b)one or more modified nucleotides within five nucleotides from said3′-end, or (c) both (a) and (b).
 13. The crRNA molecule of claim 12wherein said crRNA molecule comprises: (a) one modified nucleotide atthe first nucleotide of said 5′-end, or (b) at least one modifiednucleotide within two nucleotides from said 3′-end, or (c) both (a) and(b).
 14. The crRNA molecule of claim 12 wherein said modified nucleotidecomprises a modified internucleotide linkage or a modified terminalphosphate group selected from an alkylphosphonate, aphosphonocarboxylate, a phosphonoacetate, a boranophosphonate, aphosphorothioate, a phosphonothioacetate, and a phosphorothioate group.15. The crRNA molecule of claim 12 wherein said crRNA comprises amodified internucleotide linkage or a modified terminal phosphate groupselected from a phosphonocarboxylate, a phosphonoacetate, and aphosphonothioacetate group.
 16. The crRNA molecule of claims 12, whereinsaid one or more modifications are independently selected from a2′-deoxy, 2′-O-methyl, 3′-phosphorothioate, 3′-phosphonoacetate (PACE),3′-phosphonothioacetate (thioPACE), and a combination thereof.
 17. ThecrRNA molecule of claim 12 wherein said one or more modified nucleotidescomprise a 2′-O-methyl-3′-phosphorothioate nucleotide.
 18. The crRNAmolecule of claim 12 wherein said one or more modified nucleotidescomprise a 2′-O-methyl-3′-phosphonoacetate nucleotide or a2′-O-methyl-3′-phosphonothioacetate nucleotide or a combination thereof.19. The crRNA molecule of claim 13 wherein said one modified nucleotideis independently selected from a 2′-O-methyl nucleotide, 2′-O-methyl-3′-phosphorothioate nucleotide, 3′-phosphorioacetate nucleotide and2′-O-methyl-3′-phosphonothioacetate nucleotide.
 20. The crRNA moleculeof claim 15 wherein, said modified nucleotides are each independentlyselected from a 2′-O-methyl nucleotide, 2′-O-methyl-3′-phosphorothioatenucleotide, 2′-O-methyl-3′-phosphonoacetate nucleotide and2′-O-methyl-3′-phosphonothioacetate nucleotide.
 21. A method formodifying a DNA sequence, regulating the expression of a gene ofinterest, or cleaving a target polynucleotide, the method comprising:selecting at least one target polynucleotide; providing at least onesynthetic guide RNA of claim 1; forming a gRNA:Cas protein complexcomprising a Cas protein and the synthetic guide RNA; and contacting thetarget polynucleotide with the gRNA:Cas protein complex; wherein saidGas protein is provided as a protein or as a polynucleotide encodingsaid Cas protein.
 22. The method of claim 21, wherein said guide RNAcomprises: (a) one modified nucleotide at the first nucleotide of said5′-end, or (b) at least one modified nucleotide within two nucleotidesfrom said 3′-end, or (c) both (a) and (b).
 23. The method of claim 21wherein said modified nucleotide comprises a modified internucleotidelinkage or a modified terminal phosphate group selected from analkylphosphonate, a phosphonocarboxylate, a phosphonoacetate, aboranophosphonate, a phosphorothioate, a phosphonothioacetate, and aphosphorothioate group.
 24. The method of claim 21 wherein said one ormore modified nucleotides comprise a 2′-O-methyl-3′-phosphorothioatenucleotide.
 25. The method of claim 21 wherein said one or more modifiednucleotides comprise a 2′-O-methyl-3′-phosphonoacetate nucleotide or a2′-O-methyl-3′-phosphonothioacetate nucleotide or a combination thereof.26. The method of claim 22 wherein said modified nucleotides are eachindependently selected from a 2′-O-methyl nucleotide,2′-O-methyl-3′-phosphorothioate nucleotide,2′-O-methyl-3′-phosphonoacetate nucleotide and2′-O-methyl-3′-phosphonothioacetate nucleotide.
 27. The method of claim21 wherein said forming of said gRNA:Cas protein complex is performedoutside or inside a cell and said contacting is performed in a cell. 28.The method of claim 21 wherein said Cas protein is a Cas9 protein. 29.The method of claim 21 wherein said forming is performed outside a cell.30. A set or a library comprising at least two guide RNAs of claim 1.